Cloning and characterizing of genes associated with long-term memory

ABSTRACT

A method of regulating long term memory is disclosed. Also disclosed is isolated DNA encoding a cyclic 3&#39;,5&#39;-adenosine monophosphate responsive transcriptional activator, isolated DNA encoding an antagonist of cyclic 3&#39;,5&#39;-adenosine monophosphate-inducible transcription, isolated DNA encoding an enhancer-specific activator, and isolated DNA encoding a nitric oxide synthase. A method for assessing the effect of a drug on long term memory formation is also disclosed.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No. 1RO1 HD32245-01 awarded by the National Institutes of Health. The United States government has certain rights in this invention.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 08/319,866, filed Oct. 7, 1994, which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Activation of the cyclic 3',5'-adenosine monophosphate (cAMP) signal transduction pathway can have long-lasting global consequences through its influence on the expression of specific genes. This is true for simple organisms as well as mammals, where many of the known cAMP-responsive genes can have important neural and endocrine roles. Additional information regarding activation of this pathway would be useful, particularly as this activation pertains to the ability of animals to remember activities or events.

SUMMARY OF THE INVENTION

The present invention is based on Applicants' discovery of the dCREB1 and dCREB2 genes. The present invention is further based on Applicants' discovery that the Drosophila CREB2 gene codes for proteins of opposite functions. One isoform (e.g., dCREB2-a) encodes a cyclic 3',5'-adenosine monophosphate (cAMP)-responsive transcriptional activator. Another isoform (e.g., dCREB2-b) codes for an antagonist which blocks the activity of the activator.

When the blocking form is placed under the control of the heat-shock promoter, and transgenic flies are made, a brief shift in temperature induces the synthesis of the blocker in the transgenic fly. This induction of the blocker (also referred to herein as the repressor) specifically disrupts long-term, protein synthesis dependent memory of an odor-avoidance behavioral paradigm.

As a result of Applicants' discovery, a method is herein provided to regulate long term memory in an animal. The method of regulating long term memory described herein comprises inducing expression of a dCREB2 gene or a fragment thereof in the animal.

The dCREB2 gene encodes several isoforms. Examples of an isoform encoded by the dCREB2 gene are dCREB2-a, dCREB2-b, dCREB2-c, dCREB2-d, dCREB2-q, dCREB2-r and dCREB2-s.

The isoforms encoded by the dCREB2 gene include cAMP-responsive activator isoforms and antagonistic blocker (or repressor) isoforms of the activator isoforms. Cyclic AMP responsive activator isoforms can function as a cAMP-responsive activator of transcription. Antagonistic repressors can act as a blocker of activators. An example of a cAMP-responsive activator isoform is dCREB2-a. An example of an antagonistic repressor (or blocker) isoform is dCREB2-b. The terms blocker and repressor are used interchangeably herein.

In one embodiment of the invention, the dCREB-2 gene encodes a cAMP-responsive activator isoform and inducing said gene results in the potentiation of long term memory.

Alternatively, inducing the dCREB2 gene encoding a cAMP-responsive activator isoform activates the production of a protein which is necessary for the formation of long term memory.

In another embodiment of the invention, the dCREB2 gene encodes a repressor isoform and inducing said gene results in the blocking of long term memory.

A further embodiment of the invention relates to a method of regulating long term memory in an animal comprising inducing repressor and activator isoforms of dCREB2 wherein long term memory is potentiated in the animal when the net amount of functional activator (ΔC) is greater than zero.

The invention also relates to a method of identifying a substance capable of affecting long term memory in an animal comprising the determination that said substance alters the induction or activity of repressor and activator isoforms of dCREB2 from normal in the animal.

As referred to herein, an activator isoform includes dCREB2-a and functional fragments thereof and a repressor isoform includes dCREB2-b and functional fragments thereof.

Other embodiments of the invention relate to a method of enhancing long term memory formation in an animal comprising increasing the level of activator homodimer from normal, decreasing the level of activator-repressor heterodimer from normal, or decreasing the level of repressor homodimer from normal in the animal.

Still another embodiment of the invention relates to a method of identifying a substance capable of affecting long term memory in an animal comprising the determination that said substance alters activator homodimer, activator-repressor heterodimer and/or repressor homodimer formation from normal in the animal.

As referred to herein, an activator homodimer includes the dCREB2a homodimer, an activator-repressor heterodimer includes the dCREB2a-dCREB2b heterodimer, and a repressor homodimer includes the dCREB2b homodimer.

A further embodiment of the invention relates to isolated DNA encoding a cAMP responsive transcriptional activator. Such a cAMP responsive transcriptional activator can be encoded by a Drosophila dCREB2 gene or by homologues or functional fragments thereof. For example, a cAMP responsive transcriptional activator can be encoded by the dCREB2 gene which codes for dCREB2-a or by a gene encoded by the sequences presented herein.

Still another embodiment of the invention relates to isolated DNA encoding an antagonist of cAMP-inducible transcription. Such an antagonist of cAMP-inducible transcription can be encoded by a Drosophila dCREB2 gene or by homologues or functional fragments thereof. For example, an antagonist of cAMP-inducible transcription can be encoded by the dCREB2 gene which codes for dCREB2-b.

Another embodiment of the invention relates to isolated DNA which encodes a Drosophila dCREB2 gene or functional fragments thereof.

A further embodiment of the invention relates to isolated DNA encoding an enhancer-specific activator. Such an enhancer-specific activator can be encoded by a Drosophila dCREB1 gene or by homologues or functional fragments thereof.

Another embodiment of the invention relates to isolated DNA encoding a nitric oxide synthase of Drosophila (DNOS). Such DNA can encode a DNOS of neuronal locus. The DNOS encoded can contain, for example, putative heme, calmodulin, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate, in its reduced form, (NADPH) binding site domains.

A further embodiment of the invention relates to a method for assessing the effect of a drug on long term memory formation comprising administering the drug to Drosophila, subjecting the Drosophila to classical conditioning to at least one odorant and electrical shock, and assessing the performance index of the classical conditioning, wherein the effect of the drug occurs when it alters the performance index from normal. The drug can affect long term memory formation by, for example, altering the induction or activity of repressor and activator isoforms of dCREB2.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts the DNA sequence (SEQ ID NO.: 1) and predicted amino acid sequence (SEQ ID NO.: 2) of the dCREB2-a coding region. The basic region and leucine zipper domains are indicated by solid and broken bold underlining, respectively; positively-charged residues in the basic region are circled; periodic leucines in the zipper motif are boxed; glutamines in the activation domain are underlined; the short amino acid motif with target sites for kinases, starting at residue 227, is indicated by a bold outline; and sequences specified by alternatively-spliced exons 2, 4 and 6 are shaded.

FIG. 1B depicts the amino acid sequences of the bZIP domains of dCREB2 (SEQ ID NO.: 3), mammalian CREB (SEQ ID NO.: 4), CREM (SEQ ID NO.: 5) and ATF-1 (SEQ ID NO.: 6). Differences between dCREB2 and CREB are boxed.

FIG. 2 is a schematic diagram of dCREB2 isoforms with the exon boundaries defined with respect to dCREB2-a. Diagram is not drawn to scale.

FIG. 3 is a bar graph representation of results showing pKA-responsive transcriptional activation by dCREB2-a.

FIG. 4 is a bar graph representation of results showing the transcriptional effect of dCREB2-b and a mutant variant on pKA-responsive activation by dCREB2-a.

FIG. 5 depicts the DNA sequence (SEQ ID NO.: 7) and predicted amino acid sequence (SEQ ID NO.: 8) of the dCREB1 coding region. The basic region and leucine zipper domains are indicated by solid and broken bold underlining, respectively; positively-charged residues in the basic region are circled; periodic leucines of the zipper motif are boxed; and in the acid-rich region of the activation domain, negatively-charged amino acids are underlined and proline residues are indicated by diamonds.

FIG. 6 is a bar graph representation of results showing transcriptional activation of a CRE reporter gene by dCREB1 in Drosophila Schneider L2 cell culture.

FIG. 7A is a photomicrograph of a Northern blot depicting the effect of heat shock induction on dCREB2-b expression: wt=wildtype flies; CREB=17-2 transgenic flies; lanes 1-2: no heat shock; lanes 2-3: immediately after heat shock; lanes 5-6: three hours after heat shock.

FIG. 7B is a photograph of a Western blot depicting the effect of heat shock induction on dCREB2-b protein production: wt=wildtype flies; CREB=17-2 transgenic flies; lanes 1-2: no heat shock; lanes 2-3: immediately after heat shock; lanes 5-6: one hour after heat shock; lanes 7-8: three hours after heat shock; lanes 9-10: 9 hours after heat shock; lanes 11-12: 24 hours after heat shock.

FIG. 7C is a photograph of a Western blot depicting the effect of heat shock induction on dCREB2 and dCREB2-mLZ (a mutated dCREB2-b) protein production: wt=17-2 transgenic flies (expressing wildtype blocker, dCREB2-b); m=A2-2 transgenic flies (expressing mutant blocker, dCREB2-mLZ); lanes 1-2: no heat shock; lanes 3-4: immediately after heat shock; lanes 5-6: three hours after heat shock; lanes 7-8: six hours after heat shock.

FIG. 8 is a bar graph representation of results showing the effect of cycloheximide (CXM) feeding, before or after spaced or massed training, on one-day memory retention: striped bars=+CXM; white bars=-CXM.

FIG. 9A is a bar graph representation of results showing the effect of heat shock induction on one-day memory retention in wildtype (Can-S) flies and hs-dCREB2-b transgenic (17-2) flies given spaced or massed training: white bars=wildtype (Can-S) flies; striped bars: hs-dCREB2-b transgenic (17-2) flies; hs=heat shock.

FIG. 9B is a bar graph representation of results showing the effect of heat shock induction on one-day memory retention in wildtype (Can-S) flies or hs-dCREB2-b transgenic (M11-1) flies given spaced or massed training: white bars=wildtype (Can-S) flies; striped bars: hs-dCREB2-b transgenic (M11-1) flies; hs=heat shock.

FIG. 9C is a bar graph representation of results showing the effect of heat shock induction on learning in wildtype (Can-S) flies and hs-dCREB2-b transgenic (17-2) flies given spaced or massed training: white bars=wildtype (Can-S) flies; striped bars: hs-dCREB2-b transgenic (17-2) flies; hs=heat shock.

FIG. 10 is a bar graph representation of results showing the effect of heat shock induction on one-day memory retention in wildtype [w(isoCJ1)] flies, hs-dCREB2-b transgenic (17-2) flies, and mutant hs-dCREB2-mLZ transgenic (A2-2) flies given spaced training: forward striped bars=wildtype [w(isoCJ1)] flies; back striped bars=hs-dCREB2-b transgenic (17-2) flies; white bars=mutant hs-dCREB2-mLZ transgenic (A2-2) flies; hs=heat shock.

FIG. 11 is a bar graph representation of results showing the effect of heat shock induction on seven-day memory retention (long term memory) in wildtype (Can-S) flies and hs-dCREB2-b transgenic (17-2) flies given spaced training: white bars=wildtype (Can-S) flies; striped bars=hs-dCREB2-b transgenic (17-2) flies; hs=heat shock.

FIG. 12 is a bar graph representation of results showing the effect of heat shock induction on one-day memory retention in hs-dCREB2-b transgenic (17-2) flies, radish mutant flies, and radish hs-dCREB2-b double mutant (rsh;17-2) flies given spaced training: hs=heat shock; white bars=-hs; striped bars=+hs.

FIG. 13A is a graphic representation of results showing the effect of repeated training sessions on seven-day memory retention (long term memory) in wildtype (Can-S) flies with long term memory as a function of the number of training sessions indicated by open circles and a negative accelerating exponential Gompertz (growth) function fit to the individual performance indexes (PIs) using a nonlinear iterative Least squares method indicated by the solid line.

FIG. 13B is a graphic representation of results showing the effect of the rest interval between each training session on seven-day memory retention (long term memory) in wildtype (Can-S) flies with long term memory as a function of the rest interval indicated by open circles and a negative accelerating exponential Gompertz (growth) function fit to the individual performance indexes (PIs) using a nonlinear iterative least squares method indicated by the solid line.

FIG. 14 depicts a conceptual model of a molecular switch for the formation of long term memory based on differential regulation of CREB isoforms with opposing functions with ΔC indicating the net effect of CREB activators.

FIG. 15A is a bar graph representation of results showing the effect of 48 massed training sessions (48× massed) or 10 spaced training sessions with a 15-minute rest interval (10× spaced) on seven-day memory retention in wildtype (Can-S) flies.

FIG. 15B is a bar graph representation of results showing the effect of one (1×), two (2×) or ten (10×) massed training sessions, three hours after heat-shock induction of the transgene (induced) or in the absence of heat-shock (uninduced), on seven-day memory retention in wildtype (Can-S) flies, hsp-dCREB2-a transgenic (C28) flies, and hsp-dCREB2-a transgenic (C30) flies: forward striped bars=wildtype (Can-S) flies; back striped bars=hsp-dCREB2-a transgenic (C28) flies; and white bars=hsp-dCREB2-a transgenic (C30) flies.

FIG. 15C is a bar graph representation of results showing responses three hours after heat shock in wildtype (Can-S) flies and hsp-dCREB2-a transgenic (C28) flies to odors, either octanol (OCT) or methylcyclohexanol (MCH), or to shock (60 V DC): forward striped bars=wildtype (Can-S) flies; back striped bars=hsp-dCREB2-a transgenic (C28) flies.

FIGS. 16A-16C depict the deduced amino acid sequences of DNOS and mammalian NOSs with amino acid numbering starting at the first methionine in each open reading frame (ORF), putative binding domains for cofactors (overlined) demarcated as in previously published reports on mammalian NOSs, and amino acids which have been proposed as contacts with FAD and NADPH based on crystal structure of the ferrodoxin NADP+reductase (40) conserved at equivalent positions (bullet points): DNOS, Drosophila NOS (SEQ ID NO.: 9); RNNOS, rat neuronal NOS (SEQ ID NO.: 10); BENOS, bovine endothelial NOS (SEQ ID NO.: 11); MMNOS, mouse macrophage NOS (SEQ ID NO.: 12). Sequence alignment and secondary structure predictions were performed by Geneworks 2.3 (IntelliGenetics).

FIG. 16D is a schematic diagram of the domain structure of Drosophila and mammalian NOS proteins with the proposed cofactor-binding sites for heme (H), calmodulin (CaM), flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide phosphate (NADPH) and the glutamine-rich domain (Q) in DNOS shown.

FIG. 17A is a photograph of a Western blot showing DNOS expression in 293 human embryonic kidney cells.

FIG. 17B is a bar graph representation of results showing DNOS enzyme activity measured in 293 human embryonic kidney cell extracts by conversion of ³ H-L-arginine to ³ H-L-citrulline: in the presence of exogenous Ca²⁺ or calmodulin (group B); in the presence of 1 mM EGTA without exogenous Ca²⁺ or calmodulin (group C); in the presence of 100 mM L-NAME with exogenous Ca²⁺ or calmodulin (group D).

FIG. 18A is a photomicrograph of a Northern blot showing a 5.0 kb dNOS transcipt present in Drosophila heads: H=head; B=body.

FIG. 18B is a photograph of an agarose gel stained with ethidium bromide showing the expression by the dNOS gene of two alternatively spliced mRNA species with the arrows indicating the positions of the DNA fragments of the expected sizes: the 444 bp long-form fragment and the 129 bp short-form fragment. The other bands present in the lane are artifacts from heteroduplexes that failed to denature. KB=size markers.

FIG. 18C depicts the alignment of the deduced amino acid sequence of two protein isoforms of DNOS and mouse neuronal NOS: top part shows the relation between two conceptual Drosophila NOS proteins, DNOS-1 (amino acid residues 408-427 and 513-532 of SEQ ID NO.: 9) and DNOS-2 (SEQ ID NO.: 14), corresponding to the longer and shorter RT-PCR products, respectively; the bottom part shows the relationship between the relevant regions of two protein isoforms of the mouse neuronal NOS, n-NOS-1 (amino acid residues 494-513 and 599-618; SEQ ID NO.:13 and SEQ ID NO.: 15, respectively) and n-NOS-2 (SEQ ID NO.: 16); and the numbers indicate the positions of the amino acid residues relative to the first methionine in the respective OFRs.

DETAILED DESCRIPTION OF THE INVENTION

Applicants have cloned and characterized two genes, designated dCREB2 and dCREB1, isolated through a DNA-binding expression screen of a Drosophila head cDNA library in which a probe containing three cAMP-responsive element (CRE) sites was used.

The dCREB2 gene codes for the first known cAMP-dependent protein kinase (PKA) responsive CREB/ATF transcriptional activator in Drosophila. A protein data base search showed mammalian CREB, CREM and ATF-1 gene products as homologous to dCREB2. For these reasons, dCREB2 is considered to be a member, not only of the CREB/ATF family, but of the specific cAMP-responsive CREB/CREM/ATF-1 subfamily. It is reasonable to expect that dCREB2 is involved in Drosophila processes which are analogous to those which are thought to depend on cAMP-responsive transcriptional activation in other animal systems.

Applicants have shown that the dCREB2 transcript undergoes alternative splicing. Splice products of dCREB2 were found to fall into two broad categories: one class of transcripts (dCREB2-a, -b, -c, -d) which employs alternative splicing of exons 2, 4 and 6 to produce isoforms whose protein products all have the bZIP domains attached to different versions of the activation domain and a second class of transcripts (dCREB2-q, -r, -s) which have splice sites which result in in-frame stop codons at various positions upstream of the bZIP domain. These all predict truncated activation domains without dimerization or DNA binary activity.

dCREB2-a,-b,-c and -d are splice forms that predict variants of the activation domain attached to a common basic region-leucine zipper. These alternative splice forms result in seemingly minor changes in the size and spacing of parts of the activation domain. Nevertheless, alternative splicing of the activation domain has profound effects on the functional properties of dCREB2 products. Isoform dCREB2-a produces a PKA-responsive transcriptional activator in cell culture, whereas dCREB2-b, lacking exons 2 and 6, produces a specific antagonist. This dCREB2 splicing pattern (and its functional consequences) is virtually identical to that seen in the CREM gene. Similarly located, alternatively-spliced exons in the CREM gene determine whether a particular isoform is an activator or an antagonist (14, 26).

The ability of the phosphorylation domain (KID domain) to activate in trans other constitutive transcription factors which are bound nearby could potentially transform a CREM antagonist (which contains the KID domain but is lacking an exon needed for activation) into a cAMP-responsive activator. Since the modular organization of these molecules has been conserved, dCREB2-d could have this property.

In contrast to the dCREB2 splicing variants that encode isoforms with a basic region-leucine zipper domain, the dCREB2-q, -r and -s splice forms incorporate in-frame stop codons whose predicted protein products are truncated before the bZIP region. Isoforms of this type have been identified among the products of the CREB gene (14, 64), but not the CREM gene. The function of these truncated CREB molecules is not known, but at least one such CREB mRNA is cyclically regulated in rat spermatogenesis (73).

So far, dCREB2 is the only cAMP-responsive CREB transcription factor isolated from Drosophila. Other Drosophila CREB molecules, BBF-2/dCREB-A (1,67) and dCREB1, have less homology to mammalian CREB and CREM. It may be that dCREB2 subsumes functions of both the CREB and CREM genes in Drosophila. The mammalian CREB and CREM genes are remarkably similar to one another in several respects. It has been suggested that CREB and CREM are the product of a gene duplication event (46, 62). dCREB2 has a striking degree of amino acid sequence similarity to the CREB and CREM genes in the bZIP domain. Moreover, comparison of alternative splicing patterns among CREB, CREM and dCREB2 indicates that dCREB2 generates mRNA splicing isoforms similar to exclusive products of both CREB and CREM. Taken together, the sequence information and the splicing organization suggest that dCREB2 is an ancestor of both the mammalian CREB and CREM genes.

As discussed further herein, one phenomenon in which dCREB2 might act with enduring consequences is in long-term memory. This possibility is a particularly tempting one because recent work in Aplysia indicates that a CREB factor is likely to function in long-term facilitation by inducing an "immediate early" gene (2, 13). Recent experiments with a conditionally-expressed dCREB2-b transgene indicate that it has specific effects on long-term memory in Drosophila.

The product of the second gene described herein, dCREB1, also appears to be a member of the CREB/ATF family. Gel-retardation assays indicate that it binds specifically to CREs. It has a basic region and an adjacent leucine zipper at its carboxyl end, but this domain shows limited amino acid sequence similarity to other CREB/ATF genes. The presumed transcriptional activation domain of dCREB1 is of the acid-rich variety. Furthermore, it has no consensus phosphorylation site for PKA. dCREB1 can mediate transcriptional activation from CRE-containing reporters in the Drosophila L2 cell line, but this activation is not dependent or PKA.

A recurrent finding from work on the biology of learning and memory is the central involvement of the cAMP signal transduction pathway. In Aplysia, the cAMP second-messenger system is critically involved in neural events underlying both associative and non-associative modulation of a behavioral reflex (Kandel and Schwartz, 1982; Kandel et al., 1987; Byrne et al., 1993). In Drosophila, two mutants, dunce and rutabaga, were isolated in a behavioral screen for defects in associative learning and are lesioned in genes directly involved in cAMP metabolism (Quinn, et al., 1974; Dudai et al., 1976; Byers et al., 1981; Livingstone et al., 1984; Chen et al., 1986; Levin et al., 1992). These latter observations were extended with a reverse-genetic approach using inducible transgenes expressing peptide inhibitors of cAMP-dependent protein kinase (PKA) and with analyses of mutants in the PKA catalytic subunit (Drain et al., Skoulakis et al., 1993). Recent work on mammalian long-term potentiation (LTP) also has indicated a role for cAMP in synaptic plasticity (Frey et al., 1993; Huang and Kandel, 1994; Bourtchuladze et al., 1994).

The formation of long-lasting memory in animals and of long-term facilitation in Aplysia can be disrupted by drugs that interfere with transcription or translation (Agranoff et al., 1966; Barondes and Cohen, 1968; Davis and Squire, 1984; Rosenzweig and Bennett, 1984; Montarolo et al., 1986). This suggests that memory consolidation requires de novo gene expression. Considered along with the involvement of the cAMP second-messenger pathway, this requirement for newly synthesized gene products suggests a role for cAMP-dependent gene expression in long-term memory (LTM) formation.

In mammals, a subset of genes from the CREB/ATF family are known to mediate cAMP-responsive transcription (Habener, 1990; deGroot and Sassone-Corsi, 1993). CREBs are members of the basic region-leucine zipper transcription factor superfamily (Landschulz et al., 1988). The leucine zipper domain mediates selective homo- and hetero-dimer formation among family members (Hai et al., 1989; Hai and Curran, 1991). CREB dimers bind to a conserved enhancer element (CRE) found in the upstream control region of many cAMP-responsive mammalian genes (Yamamoto et al., 1988). Some CREBs become transcriptional activators when specifically phosphorylated by PKA (Gonzalez and Montminy, 1989, Foulkes et al., 1992), while others, isoforms from the CREM gene, are functional antagonists of these PKA-responsive activators (Foulkes et al., 1991; Foulkes and Sassone-Corsi, 1992).

Work in Aplysia has shown that cAMP-responsive transcription is involved in long-term synaptic plasticity (Schacher et al., 1988; Dash et al, 1990). A primary neuronal co-culture system has been used to study facilitation of synaptic transmission between sensory and motor neurons comprising the monosynaptic component of the Aplysia gill-withdrawal reflex. Injection of oligonucleotides containing CRE sites into the nucleus of the sensory neuron specifically blocked long-term facilitation (Dash et al., 1990). This result suggests that titration of CREB activity might disrupt long-term synaptic plasticity.

Described herein is the cloning and characterization of a Drosophila CREB gene, dCREB2. This gene produces several isoforms that share overall structural homology and nearly complete amino acid identity in the basic region-leucine zipper with mammalian CREBs. The dCREB2-a isoform is a PKA-responsive transcriptional activator whereas the dCREB2-b product blocks PKA-responsive transcription by dCREB2-a in cell culture. These molecules with opposing activities are similar in function to isoforms of the mammalian CREM gene (Foulkes et al., 1991; Foulkes and Sassone-Corsi, 1992; Foulkes et al., 1992). The numerous similarities in sequence and function between dCREB2 and mammalian CREBs suggest that cAMP-responsive transcription is evolutionarily conserved.

Genetic studies of memory formation in Drosophila have revealed that the formation of a protein synthesis-dependent long-term memory (LTM) requires multiple training sessions with a rest interval between them (Tully et al. 1994). As discussed further herein, this LTM is blocked specifically by induced expression of a repressor isoform of the cAMP-responsive transcription factor CREB. Also as discussed further herein, LTM information is enhanced after induced expression of an activator form of CREB. Maximum LTM is achieved after just one training session.

To investigate the role of CREBs in long-term memory (LTM) formation in Drosophila, dominant-negative transgenic lines which express dCREB2-b under the control of a heat-shock promoter (hs-dCREB2-b) were generated. Groups of flies, which had been heat-shock induced or left uninduced, were tested for memory retention after Pavlovian olfactory learning. This acute induction regimen minimized potential complications from inappropriate expression of dCREB2-b during development and allowed a clear assessment of the effect of hs-dCREB2-b induction on memory formation.

In Drosophila, consolidated memory after olfactory learning is composed of two genetically distinct components: anesthesia-resistant memory (ARM) and long-term memory (LTM) (Tully et al., 1994). ARM decays to zero within four days after training, and formation of ARM is insensitive to the protein synthesis inhibitor cycloheximide (CXM) but is disrupted by the radish mutation (Folkers et al., 1993). In contrast, LTM shows essentially no decay over at least seven days, its formation is cycloheximide-sensitive and it is not disrupted by radish. Tully et al. (1994) employed two different training protocols involving massed and spaced sessions (Ebbinghaus, 1885; Baddeley, 1976) to dissect memory formation. The massed training procedure consists of ten consecutive training cycles with no rest interval between them, while the spaced training protocol consists of the same number of sessions but with a 15-minute rest between each. Their genetic dissection revealed that the massed protocol produced only ARM, while the spaced protocol produced memory retention composed of both ARM and LTM.

The behavioral results show that formation of LTM is completely blocked by induced expression of hs-dCREB2-b. This effect is remarkable in its behavioral specificity. ARM, a form of consolidated memory genetically distinguishable from LTM, but co-existing with it one-day after spaced training, was not affected. Learning and peripheral behaviors likewise were normal. Thus, the effect of the induced hs-dCREB2-b transgene is specific to LTM.

Induction of the mutant blocker did not affect LTM. This result, together with the molecular data which showed that induction of the wild-type blocker did not have widespread effects on transcription, suggests that the blocker is reasonably specific at the molecular level when it specifically blocks LTM. The wild-type blocker may disrupt cAMP-dependent transcription in vivo, since it can block cAMP-responsive transcription in cell culture. It is reasonable to infer that dimerization is necessary for blocker function and that the wild-type blocker could interfere with cAMP-responsive transcription either by forming heterodimers with dCREB2-a, the activator, or by forming homodimers and competing for DNA binding with homodimers of dCREB2-a. Thus, activators and repressors may form homodimers or heterodimers. It is reasonable to infer that long term memory is enhanced when the level of activator homodimer is increased from normal and/or when the level of activator-repressor heterodimer is decreased from normal and/or when the level of repressor homodimer is decreased from normal. In any case, the molecular target(s) of dCREB2-b are likely to be interesting because of the behavioral specificity of the block of LTM.

In Drosophila, consolidation of memory into long-lasting forms is subject to disruption by various agents. Tully et al. (1994) used a single-gene mutation radish and the pharmacological agent CXM to show that long-lasting memory in flies is dissectable into two components, a CXM-insensitive ARM, which is disrupted by radish, and a CXM-sensitive LTM, which is normal in radish mutants. As described herein, CREB-family members are likely to be involved in the CXM-sensitve, LTM branch of memory consolidation. The results described herein, taken together with the showing that long-term memory is dissectable into a CXM-insentive ARM and a CXM-sensitive LTM (Tully et al., 1994), show that only one functional component of consolidated memory after olfactory learning lasts longer than four days, requires de novo protein synthesis and involves CREB-family members.

Based on work in Aplysia, a model has been proposed to describe the molecular mechanism(s) underlying the transition from short-term, protein synthesis-independent to long-term, protein synthesis-dependent synaptic plasticity (Alberini et al., 1994). The present work in Drosophila on long-term memory extends this model to the whole organism. Important molecular aspects of this transition seem to involve migration of the catalytic subunit of PKA into the nucleus (Backsai et al., 1993) and subsequent phosphorylation and activation of CREB-family members (Dash et al., 1990; Kaang et al., 1992). In flies, it is likely that the endogenous dCREB2-a isoform is one of these nuclear targets. Activated dCREB2-a molecules then might transcribe other target genes, including the immediate early genes--as is apparently the case in Aplysia. (Alberini et al., 1994).

It is remarkable that the cAMP signal transduction pathway, including its nuclear components, seem to be required for memory-related functions in each of these species and behavioral tasks. Taken together with cellular analyses of a long-lasting form of LTP in hippocampal slices (Frey et al., 1993; Huang and Kandel, 1994), the emerging picture is that cAMP-responsive transcription is a conserved molecular switch involved in the consolidation of short-term memory to long-term memory. Thus, it is reasonable to infer that differential regulation of CREB isoforms serves as a molecular switch for the formation of long term memory.

A universal property of memory formation is that spaced training (repeated training sessions with a rest interval between them) produces stronger, longer-lasting memory than massed training (the same number of training sessions with no rest interval) (Ebbinghaus 1885; Hintzman 1974; Carew et al. 1972; Frost et al. 1985). This phenomenon also exists in fruit flies for a conditioned odor avoidance response (Tully et al. 1994, Tully & Quinn 1985). Genetic dissection of this long-lasting memory has revealed, however, an important difference between massed and spaced training (Tully et al. 1994). Spaced training produces two functionally independent forms of consolidated memory, ARM and LTM, while massed training produces only ARM.

As described herein, ARM and LTM differ primarily in their requirement for protein synthesis during induction (see also Tully et al. 1994). ARM is not affected when flies are fed the protein synthesis inhibitor cycloheximide (CXM) immediately before or after training, while LTM is completely blocked under the same feeding conditions. ARM in normal flies also decays away within four days after training, while LTM shows no decay for at least seven days. Thus, protein synthesis is required to induce LTM, but LTM is maintained indefinitely once formed. These latter properties of LTM have been observed throughout the animal kingdom (Davis and Squire 1984; Castellucci et al. 1989; Erber 1976; Jaffe 1980). The emerging neurobiological interpretation is that formation of LTM involves protein synthesis-dependent structural changes at relevant synapses (Greenough 1984; Buonomano and Byrne 1990; Nazif et al. 1991; Stewart 1991; Bailey and Kandel 1994). The modern molecular view is that regulation of gene expression underlies this protein synthesis-dependent effect (Goelet et al. 1986; Gall and Lauterborn 1991; Armstrong and Montminy 1993).

Why is spaced training required to induce LTM? The massed and spaced procedures both entail ten training sessions; consequently, flies receive equivalent exposure to the relevant stimuli (one odor temporally paired with electric shock and a second odor presented without shock). The only procedural difference between massed and spaced training is the rest interval between each training session. The absence of a rest interval between sessions during massed training does not appear to disrupt the memory formation process (Tully et al. 1994). The level of initial learning assayed immediately after massed training is similar to that after spaced training. In addition, ARM levels are similar after both training procedures. Thus, the presence of a rest interval during spaced training seems crucial to the induction of LTM.

To investigate the temporal kinetics of this rest interval in relation to the formation of LTM (FIGS. 13A and 13B), it was first established that the usual ten sessions of spaced training produced maximal 7-day memory retention (7-day retention is composed solely of LTM, since ARM decays to zero within four days; Tully et al. 1994).

FIG. 13A shows that 15 or 20 training sessions did not improve memory retention. Thus, ten spaced training sessions produces maximal, asymptotic levels of LTM.

LTM as a function of the length of the rest interval between 10 spaced training sessions was then assessed. FIG. 13B reveals a continuous increase in LTM from a 0-min rest interval (massed training) to a 10-min rest interval, at which time LTM levels reach maximum. Longer rest intervals yielded similar memory scores. These observations of LTM formation suggest an underlying biological process, which changes quantitatively during the rest interval between sessions and which accumulates over repeated training sessions.

In transgenic flies, the formation of LTM, but not ARM or any other aspect of learning or memory, is disrupted by induced expression of a repressor form of the cAMP-responsive transcription factor CREB (Example 4). Mutating two amino acids in the "leucine zipper" dimerization domain of this CREB repressor was sufficient to prevent the dominant-negative effect on LTM. Thus, indication of LTM is not only protein synthesis-dependent but also is CREB-dependent. Stated more generally, CREB function is involved specifically in a form of a memory that is induced only by spaced training. This observation was particularly intriguing in light of the molecular nature of CREB.

In Drosophila, transcriptional and/or post-translational regulation of dCREB2 yields several mRNA isoforms. Transient transfection assays in mammalian F9 cells have demonstrated that one of these isoforms (CREB2-a) functions as a cAMP-responsive activator of transcription, while a second isoform (CREB2-b) acts as an antagonistic repressor of the activator (Example 1; cf. Habener 1990; Foulkes and Sassone-Corsi 1992). (This repressor isoform was used previously to generate the inducible transgene mentioned above.) The existence of different CREB isoforms with opposing functions suggested an explanation for the requirement of multiple training sessions with a rest interval between them for the formation of LTM.

In its simplest form, this model (Example 7; FIG. 14) supposes that cAMP-dependent protein kinase (PKA), activated during training, induces the synthesis and/or function of both CREB activator and repressor isoforms (cf. Yamamoto et al. 1988; Backsai et al. 1993). Immediately after training, enough CREB repressor exists to block the ability of CREB activator to induce downstream events. Then, CREB repressor isoforms are inactivated faster than CREB activator isoforms. In this manner, the net amount of functional activator (ΔC=CREB2a-CREB2b) increases during a rest interval and then accumulates over repeated training sessions (with a rest interval) to induce further the downstream targets involved with the formation of LTM (Montarolo et al. 1986; Kaang et al. 1993).

This model leads to three predictions, which have been confirmed. First, if the functional difference between CREB activator and repressor isoforms is zero (ΔC=0) immediately after one training session, then additional massed training sessions should never yield LTM. FIG. 15A shows that 48 massed training sessions, rather than the usual 10, still does not produce any 7-day memory retention. Second, if the amount of CREB repressor is increased experimentally, ΔC will be negative immediately after training (ΔC<0). Then, enough CREB repressor may not decay during a rest interval to free enough CREB activator for induction of LTM. This has been shown to be the case for spaced training (15-min rest interval) after inducing the expression of a hsp-dCREB2-b (repressor) transgene three hours before training (Example 4). Third, if the amount of CREB activator is increased experimentally, ΔC will be positive immediately after training (ΔC>0). This effect, then, should eliminate or reduce the rest interval required to induce LTM. FIG. 15B shows the results from recent experiments in which the expression of a hsp-dCREB2-a (activator) transgene was induced three hours before training. In these transgenic flies, massed training produced maximal LTM. This effect appeared not to arise trivially, since olfactory acuity, shock reactivity (FIG. 15C) and initial learning were normal in transgenic flies after heat shock-induction. Thus, the requirement for a rest interval between training sessions to induce LTM specifically was eliminated.

FIG. 15B also shows that maximal LTM occurred in induced hsp-dCREB2-a transgenic flies after just one training session. The usual requirement for additional training to form a strong, long-lasting memory was no longer necessary. Thus, induced overexpression of a CREB activator has produced in otherwise normal flies, the functional equivalent of a "photographic" memory. This result indicates that the amount of CREB activator present during training--rather than the amount of activated PKA that reaches CREB in the nucleus, for instance (cf. Backsai et al. 1993; Kaang et al. 1993; Frank and Greenberg 1994)--is the rate-limiting step of LTM formation. Taken together, results from these experiments support the notion that the opposing functions of CREB activators and repressors act as a "molecular switch" (cf. Foulkes et al. 1992) to determine the parameters of extended training (number of training sessions and rest interval between them) required to form maximum LTM.

To date, seven different dCREB2 RNA isoforms have been identified, and more are hypothesized to exist. Each may be regulated differentially at transcriptional (Meyer et al. 1993) and/or translation levels before or during LTM formation. In addition, different combinations of CREB isoforms may exist in different (neuronal) cell types. Consequently, many different combinations of activator and repressor molecules are possible. From this perspective, the notions that all activators and repressors are induced during a training session or that all repressors inactivate faster than activators (see above) need not be true. Instead, the model requires only that ΔC (the net function of activators and repressors) is less than or equal to zero immediately after training and the increases with time (rest interval).

Theoretically, particular combinations of activator and repressor molecules in the relevant neuron(s) should determine the rest interval and/or number of training sessions necessary to produce maximum LTM for any particular task or species. Thus, the molecular identification and biochemical characterization of each CREB activator and repressor isoform used during LTM formation in fruit flies is the next major step toward establishing the validity of our proposed model. Similar experiments in other species may establish its generality.

CREB certainly is not involved exclusively with LTM. The dCREB2 gene, for instance, is expressed in all fruit fly cells and probably acts to regulate several cellular events (Foulkes et al. 1992).

So, what defines the specificity of its effects on LTM? Specificity most likely resides with the neuronal circuitry involved with a particular learning task. For olfactory learning in fruit flies, for instance, CREB probably is modulated via the cAMP second messenger pathway. Genetic disruptions of other components of this pathway are known to affect olfactory learning and memory (Livingstone et al. 1984; Drain et al. 1991; Levin et al. 1992; Skoulakis et al. 1993; Qiu and Davis 1993). Presumably, the stimuli used during conditioning (training) stimulate the underlying neuronal circuits. The cAMP pathway is activated in (some) neurons participating in the circuit, and CREB-dependent regulation of gene expression ensues in the "memory cells". This neurobiological perspective potentially will be established in Drosophila by identifying the neurons in which LTM-specific CREB function resides. Experiments using a neuronal co-culture system in Aplysia already have contributed significantly to this issue (Alberini et al. 1994 and references therein).

The involvement of CREB in memory, or in the structural changes of neurons which underlie memory in vivo, also has been implicated in mollusks (Dash et al. 1990; Alberini et al. 1994) and in mice (Bourtchuladze et al. 1994). Ample evidence also exists for the involvement of the cAMP second messenger pathway in associative learning in Aplysia (Kandel et al. 1987; Byrne et al. 1993) and in rat hippocampal long-term potentiation (LTP), a cellular model of associative learning in vertebrates (Frey et al. 1993; Huang and Kandel 1994). Finally, cellular and biochemical experiments have suggested that CREB function may be modulated by other second messenger pathways (Dash et al. 1991); Ginty et al. 1994; deGroot et al. 1994). These observation suggest that CREB might act as a molecular switch for LTM in many species and tasks.

Finally, why might the formation of LTM require a molecular switch? Many associative events occur only once in an animal's lifetime. Forming long-term memories of such events would be unnecessary and if not counterproductive. Instead, discrete events experienced repeatedly are worth remembering. In essence, a recurring event comprises a relevant signal above the noise of one-time events. Teleologically, then, the molecular switch may act as an information filter to ensure that only discrete but recurring events are remembered. Such a mechanism would serve efficiently to tailor an individual's behavioral repertoire to its unique environment.

The present invention also relates to isolated DNA having sequences which encode (1) a cyclic 3',5'-adenosine monophosphate (cAMP) responsive transcriptional activator, or a functional fragment thereof, or (2) an antagonist of a cAMP responsive transcriptional activator, or a functional fragment thereof, or (3) both an activator and an antagonist, or functional fragments thereof of both.

The invention relates to isolated DNA having sequences which encode Drosophila dCREB2 isoforms, or functional analogues of a dCREB2 isoform. As referred to herein, a functional analogue of a dCREB2 isoform comprises at least one function characteristic of a Drosophila dCREB2 isoform, such as a cAMP-responsive transcriptional activator function and/or an antagonistic repressor of the cAMP activator function. These functions (i.e., the capacity to mediate PKA-responsive transcription) may be detected by standard assays (e.g., assays which monitor for CREB-dependent activation). For example, assays in F9 cells have been used extensively to study CREB-dependent activation because their endogenous cAMP-responsive system is inactive (28, 49, 50).

The invention further relates to isolated DNA having sequences which encode a Drosophila dCREB2 gene or a functional fragment thereof. Isolated DNA meeting these criteria comprise nucleic acids having sequences identical to sequences of naturally occurring Drosophila dCREB2 and portions thereof, or variants of the naturally occurring sequences. Such variants include mutants differing by the addition, deletion or substitution of one or more nucleic acids.

The invention relates to isolated DNA that are characterized by (1) their ability to hybridize to a nucleic acid having the DNA sequence in FIG. 1A (SEQ ID NO.: 1) or its complement, or (2) by their ability to encode a polypeptide of the amino acid sequence in FIG. 1A (SEQ ID NO.: 2) or functional equivalents thereof (i.e., a polypeptide which functions as a cAMP responsive transcriptional activator), or (3) by both characteristics. In one embodiment, the percent amino acid sequence similarity between the polypeptide of FIG. 1A and functional equivalents thereof is at least about 55% (≧55%). In a preferred embodiment, functional equivalents of the polypeptide of FIG. 1A share at least about 60% sequence similarity with the polypeptide of FIG. 1A. More preferably, the percent amino acid sequence similarity between the polypeptide of FIG. 1A and functional equivalents thereof is at least about 70%, and still more preferably, at least about 75%. Isolated nucleic acids meeting these criteria comprise nucleic acids having sequences homologous to sequences of mammalian CREB, CREM and ATF-1 gene products. Isolated nucleic acids meeting these criteria also comprise nucleic acids having sequences identical to sequences of naturally occurring dCREB2 or portions thereof, or variants of the naturally occurring sequences. Such variants include mutants differing by the addition, deletion or substitution of one or more residues, modified nucleic acids in which one or more residues is modified (e.g., DNA or RNA analogs), and mutants comprising one or more modified residues.

Such nucleic acids can be detected and isolated under high stringency conditions or moderate stringency conditions, for example. "High stringency conditions" and "moderate stringency conditions" for nucleic acid hybridizations are explained on pages 2.10.1-2.10.16 (see particularly 2.10.8-11) and pages 6.3.1-6 in Current Protocols in Molecular Biology (Ausubel, F. M. et al., eds, Vol. 1, Suppl. 26, 1991), the teachings of which are incorporated herein by reference. Factors such as probe length, base composition, percent mismatch between the hybridizing sequences, temperature and ionic strength influence the stability of nucleic acid hybrids. Thus, high or moderate stringency conditions can be deterimined empirically, depending in part upon the characteristics of the known DNA to which other unknown nucleic acids are being compared for homology.

Isolated nucleic acids that are characterized by their ability to hybridize to a nucleic acid having the sequence in FIG. 1A or its complement (e.g., under high or moderate stringency conditions) may further encode a protein or polypeptide which functions as a cAMP responsive transcriptional activator.

The present invention also relates to isolated DNA having sequences which encode an enhancer-specific activator, or a functional fragment thereof.

The invention further relates to isolated DNA having sequences which encode a Drosophila dCREB1 gene or a functional fragment thereof. Isolated DNA meeting these criteria comprise nucleic acids having sequences identical to sequences of naturally occurring Drosophila dCREB1 and portions thereof, or variants of the naturally occurring sequences. Such variants include mutants differing by the addition, deletion or substitution of one or more nucleic acids.

The invention further relates to isolated DNA that are characterized by (1) their ability to hybridize to a nucleic acid having the DNA sequence in FIG. 5 (SEQ ID NO.: 7) or its complement, or (2) by their ability to encode a polypeptide of the amino acid sequence in FIG. 5 (SEQ ID NO.: 8), or by both characteristics. Isolated DNA meeting these criteria also comprise nucleic acids having sequences identical to sequences of naturally occurring dCREB1 or portions thereof, or variants of the naturally occurring sequences. Such variants include mutants differing by the addition, deletion or substitution of one or more residues, modified nucleic acids in which one or more residues is modified (e.g., DNA or RNA analogs), and mutants comprising one or more modified residues.

Such nucleic acids can be detected and isolated under high stringency conditions or moderate stringency conditions as described above, for example.

Fragments of the isolated DNA which code for polypeptides having a certain function can be identified and isolated by, for example, the method of Jasin, M., et al., U.S. Pat. No. 4,952,501.

Nitric Oxide in Invertebrates: Drosophila dNOS Gene Codes for a Ca²⁺ /Calmodulin-Dependent Nitric Oxide Synthase

Nitric oxide (NO) is a gaseous mediator of a wide variety of biological processes in mammalian organisms. Applicants have cloned the Drosophila gene, dATOS, coding for a Ca²⁺ /calmodulin-dependent nitric oxide synthase (NOS). Presence of a functional NOS gene in Drosophila provides conclusive evidence that invertebrates synthesize NO and presumably use it as a messenger molecule. Furthermore, conservation of an alternative RNA splicing pattern between dNOS and vertebrate neuronal NOS, suggests broader functional homology in biochemistry and/or regulation of NOS.

NO is synthesized by nitric oxide synthases (NOSs) during conversion of L-arginine to L-citrulline (1). Biochemical characterization of NOSs has distinguished two general classes: (i) constitutive, dependent on exogenous Ca²⁺ and calmodulin and (ii) inducible, independent of exogenous Ca²⁺ and calmodulin. Analyses of cDNA clones have identified at least three distinct NOS genes in mammals (2, 3, 4, 5) neuronal, endothelial and macrophage, the former two of which are constitutive and the latter of which is inducible. The nomenclature for these different isoforms used here is historical, as it is clear now that one or more isoforms can be present in the same tissues (6).

As a diffusible free-radical gas, NO is a multifunctional messenger affecting many aspects of mammalian physiology [for reviews, see (7)]. NO originally was identified as an endothelium-derived relaxing factor responsible for regulation of vascular tone (8) and as a factor involved with macrophage-mediated cytotoxicity (9). Since NO has been implicated in several physiological processes including inhibition of platelet aggregation, promotion of inflammation, inhibition of lymphocyte proliferation and regulation of microcirculation in kidney (10). More recently, NO also has been shown to play a role in cell--cell interactions in mammalian central and peripheral nervous systems--in regulating neurotransmitter release, modulation of NMDA receptor-channel functions, neurotoxicity, nonadrenergic noncholonergic intestinal relaxation (11) and activity-dependent regulation of neuronal gene expression (12). Recent reports of NO function in synaptogenesis and in apoptosis during development of the rat CNS (13) suggest that NO regulates activity-dependent mechanism(s) underlying the organization of fine-structure in the cortex (14). NO also appears to be involved with long-term potentiation in hippocampus and long-term depression in cerebellum, two forms of synaptic plasticity that may underlie behavioral plasticity (15). Consistent with these cellular studies, inhibition of NOS activity has been shown to disrupt learning and memory (16).

Many of the above conclusions are based on pharmacological studies using inhibitors of nitric oxide synthases or donors of NO. Interpretations of such studies usually are limited because the drugs interact with more than one target and they cannot be delivered to specific sites. A molecular genetic approach can overcome these problems, however, by disrupting a specific gene, the product of which may be one of the drug's targets. Recently, such an approach has been attempted in mice via generation of a knock-out mutation of the neuronal NOS (nNOS) (17). While nNOS mutants appeared fully viable and fertile, minor defects in stomach morphology and hippocampal long-term potentiation were detected (17, 18). Moreover, some NOS enzymatic activity still was present in certain regions of the brain, suggesting a role for other NOS genes in the CNS. While yielding some relevant information about one specific component of NO function, this nNOS disruption existed throughout development. Consequently, functional defects of NOS disruption in adults could not be resolved adequately from structural defects arising during development. Genetic tools exist in Drosophila, in contrast, to limit disruptions of gene functions temporally or spatially.

To identify candidate Drosophila NOS homologs, a fragment of the rat neuronal NOS CDNA (2) was hybridized at low stringency to a phage library of the Drosophila genome as described in Example 11 (19). The rat cDNA fragment encoded the binding domains of FAD and NADPH (amino acids 979-1408 of SEQ ID NO.: 11), which are cofactors required for NOS activity, and therefore were expected to be conserved in fruit flies. Several Drosophila genomic clones were identified with the rat probe and classified into eight contigs. Sequence analysis of three restriction fragments from these genomic clones revealed one (2.4R) with high homology to mammalian NOSs. The deduced amino acid sequence of the ORF encoded within the 2.4R fragment indicated 40% identity to the rat neuronal NOS and binding sites for FAD and NADPH.

The 2.4R DNA fragment then was used to probe a Drosophila adult head cDNA library as described in Example 11 (19), and eight clones were isolated. Restriction analysis indicated that all contained identical inserts and thus, defined a predominant transcript expressed by this Drosophila gene. One clone (c5.3) was sequenced in both directions. The 4491 bp cDNA contained one long ORF of 4350 bp. The methionine initiating this ORF was preceded by ACAAG which is a good match to the translation start consensus (A/CAAA/C) for Drosophila genes (20). Conceptual translation of this ORF yielded a protein of 1350 amino acids with a molecular weight of 151,842 Da.

Comparison of the amino acid sequence of this deduced Drosophila protein (DNOS) (SEQ ID NO.: 9) to sequences of mammalian NOSs revealed that DNOS is 43% identical to neuronal NOS (SEQ ID NO.: 11), 40% identical to endothelial NOS (SEQ ID NO.: 10) and 39% identical to macrophage NOS (SEQ ID NO.: 12). It also revealed similar structural motifs in DNOS (FIGS. 16A-16C). The C-terminal half of the DNOS protein contains regions of high homology corresponding to the presumptive FMN-, FAD- and NADPH-binding sites. Amino acids thought to be important for making contacts with FAD and NADPH in mammalian NOSs (2, 3, 4, 5) are conserved in DNOS. The middle section of DNOS, between residues 215 and 746 of SEQ ID NO.: 9, showed the highest similarity to mammalian NOSs: it is 61% identical to the neuronal isoform and 53% identical to endothelial and macrophage isoforms. Sequences corresponding to the proposed heme- and calmodulin-binding sites in mammalian enzymes are well-conserved in DNOS. The region located between residues 643-671 of SEQ ID NO.: 9 has the characteristics of a calmodulin-binding domain (basic, amphiphilic α-helix) (21). The amino acid sequence between these two sites is very well conserved among all four NOS proteins, suggesting the location of functionally important domains such as the arginine-binding site (3), tetrahydrobiopterine cofactor binding site or a dimerization domain. DNOS also has a PKA consensus site (22) (at Ser-287 of SEQ ID NO.: 9) in a position similar to neuronal and endothelial NOSs.

The 214 amino acid N-terminal domain of DNOS shows no obvious homology to its equivalent portion of neuronal NOS or to the much shorter N-terminal domains of endothelial and macrophage NOSs. This region of DNOS contains an almost uninterrupted homopolymeric stretch of 24 glutamine residues. Such glutamine-rich domains, found in many Drosophila and vertebrate proteins, have been implicated in protein-protein interactions regulating the activation of transcription (23). Thus, this domain of DNOS could be involved with protein--protein interactions necessary for localization and/or regulation of DNOS activity.

The above sequence comparisons suggest that a Drosophila structural homolog of a vertebrate NOS gene was identified. The order of the putative functional domains in the DNOS protein is identical to that of mammalian enzymes (FIG. 16D). Structural predictions based on several protein algorithms also indicate that general aspects of DNOS protein secondary structure (hydrophobicity plot, distribution of α-helixes and β-strands) from the putative heme-binding domain to the C-terminus are similar to those of mammalian NOSs. DNOS also does not contain a transmembrane domain, as is the case for vertebrate NOSs. In addition to these general characteristics, several aspects of DNOS structure actually render it most like neuronal NOS: (i) the overall sequence similarity, (ii) the similarity of the putative calmodulin-binding site (55% identical to the neuronal NOS vs. 45% identical to endothelial NOS or vs. 27% identical to macrophage NOS) and (iii) the large N-terminal domain. Neuronal NOS and DNOS also do not contain sites for N-terminal myristoylation, which is the case for endothelial NOS (3), nor do they have a deletion in the middle of the protein, which is the case for macrophage NOS (5).

To establish that Applicants putative DNOS protein had nitric oxide synthase activity, the dNOS cDNA was expressed in 293 human embryonic kidney cells as described in Example 12 (24), which have been used routinely in studies of mammalian NOSs (2). Protein extracts prepared from dNOS-transfected 293 cells as described in Example 12, contained a 150 kD polypeptide, which was recognized by a polyclonal antibody raised against the N-terminal domain of DNOS (FIG. 17A, lane 293+dNOS) (25). This immunoreactive polypeptide was of a size expected for DNOS and was absent from cells transfected with just the PCGN vector alone (FIG. 17A, lane 293+vector).

Extracts made from dNOS-transfected 293 cells showed significant NO synthase activity, as measured by the L-arginine to L-citrulline conversion assay as described in Example 12 (26) (0.1276±0.002 pmol/mg/min; FIG. 17B, group B). [In a parallel experiment, the specific activity of rat neuronal NOS expressed from the same vector in 293 cells was 3.0±0.02 pmol/mg/min, N=4]. DNOS activity was dependent on exogenous Ca²⁺ /calmodulin and on NADPH, two cofactors necessary for activity of constitutive mammalian NOSs (27, 28). DNOS activity was reduced 90% by the Ca²⁺ chelator EGTA (FIG. 17B, group C). Also, 500 μM N-(6-aminohexyl)-1-naphthalene-sulfonamide (W5), a calmodulin antagonist which inhibits activity of neuronal NOS (28), diminished DNOS activity to 18% (0.0222±0.001 pmol/mg/min, N=2). In the absence of exogenous NADPH, DNOS (or nNOS) activity was reduced 20% (0.1061±0.011 pmol/mg/min, N=4 for DNOS; 2.7935±0.033 pmol/mg/min, N=2 for nNOS). DNOS activity also was blocked by inhibitors of mammalian NOSs (29). N^(G) -nitro-L-arginine methyl ester (L-NAME) reduced DNOS activity 84% (FIG. 17B, group D), and 100 μM N^(G-) monomethyl-L-arginine acetate produced a complete block (0.0001±0.0002 pmol/mg/min, N=2). These enzymatic data demonstrate that DNOS is a Ca²⁺ /calmodulin-dependent nitric oxide synthase.

Northern blot analysis indicated a 5.0 kb dNOS transcript which was expressed predominantly in adult fly heads but riot bodies (FIG. 18A). More sensitive RT-PCR experiments as described in Example 13 (30), however, detected dNOS message in poly(A)⁺ RNA from fly bodies. Neuronal NOS genes from mice and humans produce two alternatively spliced transcripts, the shorter one of which yields a protein containing a 105 amino acid in-frame deletion (residues 504-608 in mouse or rat neuronal NOS) (31). RT-PCR amplification of Drosophila head mRNA produced two DNA fragments: the 444 bp fragment corresponded to vertebrate long form and the 129 bp fragment corresponded to vertebrate short form (FIG. 18B). Conceptual translation of the 129 bp sequence confirmed a splicing pattern identical to that for the nNOS gene (FIG. 18C). Presence of the short NOS isoform in Drosophila strengthens the notion that it may play an important role in NOS biochemistry.

The discovery of a NOS homolog in Drosophila provides definitive proof that invertebrates produce NO and, as suggested by recent reports, most likely use it for intercellular signaling. These data also suggest that a NOS gene was present in an ancestor common to vertebrates and arthropods, implying that NOS has existed for at least 600 million years. Thus, it is expected that NOS genes are prevalent throughout the animal kingdom.

Consistent with this view are existing histochemical data. NOS activity has been detected in several invertebrate tissue extracts: in Lymulus polyphemus (32), in the locust brain (33), in the salivary gland of Rhodnius prolixus (34) and in various tissues of Lymnaea stagnalis (35). Applications of NOS inhibitors or NO-generating substances have been shown to modulate the activity of buccal motoneurones in Lymnaea stagnalis (35) and the oscillatory dynamics of olfactory neurons in procerebral lobe of Limax maximus (36). NADPH-diaphorase staining, a relatively specific indicator of NOS protein in fixed vertebrate tissue samples (37), also has suggested the presence of NOS in Drosophila heads (38). The present molecular cloning of dNOS considerably strengthens the validity of these observations.

Sophisticated genetic analyses of NOS function are available in Drosophila. Classical genetics will allow the creation of point mutations and deletions in dNOS, resulting in full or partial loss of dNOS function. Such mutations will permit detailed studies of the role of NOS during development.

The present further invention relates to isolated DNA that are characterized by by their ability to encode a polypeptide of the amino acid sequence in FIGS. 16A1-16A3 (SEQ ID NO.: 9) or functional equivalents thereof (i.e., a polypeptide which synthesizes nitric oxide). Isolated DNA meeting this criteria comprise amino acids having sequences homologous to sequences of mammalian NOS gene products (i.e., neuronal, endothelial and macrophage NOSs). Isolated DNA meeting these criteria also comprise amino acids having sequences identical to sequences of naturally occurring ciNOS or portions thereof, or variants of the naturally occurring sequences. Such variants include mutants differing by the addition, deletion or substitution of one or more residues, modified nucleic acids in which one or more residues is modified (e.g., DNA or RNA analogs), and mutants comprising one or more modified residues.

Such nucleic acids can be detected and isolated under high stringency conditions or moderate stringency conditions, for example. "High stringency conditions" and "moderate stringency conditions" for nucleic acid hybridizations are explained on pages 2.10.1-2.10.16 (see particularly 2.10.8-11) and pages 6.3.1-6 in Current Protocols in Molecular Biology (Ausubel, F. M. et al., eds, Vol. 1, Suppl. 26, 1991), the teachings of which are incorporated herein by reference. Factors such as probe length, base composition, percent mismatch between the hybridizing sequences, temperature and ionic strength influence the stability of nucleic acid hybrids. Thus, high or moderate stringency conditions can be deterimined empirically, depending in part upon the characteristics of the known DNA to which other unknown nucleic acids are being compared for homology.

Isolated DNA that are characterized by their ability to encode a polypeptide of the amino acid sequence in FIGS. 16A-16C, encode a protein or polypeptide having at least one function of a Drosophila NOS, such as a catalytic activity (e.g., synthesis of nitric oxide) and/or binding function (e.g., putative heme, calmodulin, FMN, FAD and NADPH binding). The catalytic or binding function of a protein or polypeptide encoded by hybridizing nucleic acid may be detected by standard enzymatic assays for activity or binding (e.g., assays which monitor conversion of L-arginine to L-citrulline). Functions characteristic of dNOS may also be assessed by in vivo complementation activity or other suitable methods. Enzymatic assays, complementation tests, or other suitable methods can also be used in procedures for the identification and/or isolation of nucleic acids which encode a polypeptide having the amino acid sequence in FIGS. 16A-16C or functional equivalents thereof.

Complete citations for each of the references cited herein are provided after the examples. They are indicated in the text either by number or by author and year. List 1 is references for work relating to cloning and characterization of dCREB genes as described in Examples 1 and 2. List 2 is references for work relating to induction of dominant-negative CREB transgene and specific blocking of long-term memory, as described in Examples 3 and 4. List 3 is references for work relating to a molecular switch for long term memory, as described in Examples 5-10. List 4 is references for work relating to the dNOS gene, as described in Examples 11-13.

The present invention will now be illustrated by the following examples, which are not intended to be limiting in any way.

EXAMPLES

The following materials and methods were used in the work described in Examples 1 and 2.

Expression Cloning of dCREB1 and dCREB2

Standard protocols for expression cloning by DNA-binding (4, 66) were followed except as noted. A double-stranded, 3×CRE oligonucleotide was synthesized and cloned between the XbaI and KpnI sites of pGEM7Zf+ (Promega). The sequence of one strand of the oligonucleotide was 5' CGTCTAGATCTATGACTGAATA TGACGTAATATGACGTAATGGTACCAGATCTGGCC 3' (SEQ ID NO.: 17), with the CRE sites underlined. The oligonucleotide was excised as a BglII/HindIII fragment and labeled by filling-in the overhanging ends with Klenow fragment in the presence of [α³² P]dGTP, [α³² P]dCTP and unlabeled DATP and dTTP (4). Just prior to use, the labeled fragment was pre-absorbed to blank nitrocellulose filters to reduce background binding. All other steps were as described (4). After secondary and tertiary lifts, positive clones were subcloned into pKS+ (Stratagene) and sequences.

Gel Shift Analysis

Gel-mobility shift assays were performed as in (4) with the following modifications. The 4% polyacrylamide gel (crosslinking ratio 80:1) was cast and run using 5× Tris-glycine buffer (4) supplemented with 3 mM MgCl₂. The oligonucleotides used as the DNA probes were boiled and slowly cooled to room temperature at a concentration of 50 μg/ml in 0.1M NaCl. 50 ng of double-stranded probe was end-labeled using polynucleotide kinase in the presence of 100 μCi of [γ³² P]ATP. The double-stranded oligonucleotides were purified on a native polyacrylamide gel and used in a mobility shift assay at about 0.5 ng/reaction.

For dCREB2, the original dCREB2-b cDNA was subcloned and subjected to site-directed mutagenesis to introduce restriction sites immediately 5' and 3' of the open reading frame. This open reading frame was subcloned into the pET11A expression vector (Novagen) and used to induce expression of the protein in bacteria. The cells containing this vector were grown at 30° C. to an approximate density of 2×10⁸ /ml and heat-induced at 37° C. for 2 hours. The cells were collected by centrifugation and lysed according to (8). The crude extract was clarified by centrifugation and loaded onto a DEAE column previously equilibrated with 50 mM Tris HCl, pH 8.0, 10% sucrose, 100 mM KCl. Step elutions with increasing amounts of KCl in the same buffer were used to elute the dCREB2-b protein, which was assayed using the gel mobility-shift assay. The peak fraction was dialyzed against the loading buffer and used in the binding experiment. The specific competitor that was used was the wild-type CRE oligonucleotide. The sequence of one strand of the double-stranded oligonucleotides used in the gel shift analysis are listed. For the first two oligonucleotides, wild-type and mutant CREs are underlined.

Wild-type 3×CRE (SEQ ID NO.: 18):

5'AAATGACGTAACGGAAATGACGTAACGGAAATGACGTAACG 3';

Mutant 3×mCRE (SEQ ID NO.: 19):

5'AAATGAATTAACGGAAATGAATTAACGGAAATGAATTAACGG 3';

Nonspecific competitor #1 (SEQ ID NO.: 20):

5'TGCACGGGTTTTCGACGTTCACTGGTAGTGTCTGATGAGGCCGAAAGGCCGAAA CGCGATGCCCATAACCACCACGCTCAG 3';

Nonspecific competitor #2 (SEQ ID NO.: 21):

5'TCGACCCACAGTTTCGGGTTTTCGAGCAAGTCTGCTAGTGTCTGATGAGGCCG AAAGGCCGAAACGCGAAGCCGTATTGCACCACGCTCATCGAGAAGGC 3';

Nonspecific competitor #3 (SEQ ID NO.: 22):

5'CTAGAGCTTGCAAGCATGCTTGCAAGCAAGCATGCTTGCAAGCATGCTTG CAAGC 3';

Nonspecific competitor #4 (SEQ ID NO.: 23):

5'CTCTAGAGCGTACGCAAGCGTACGCAAGCGTACG 3'

For dCREB1, heat-induced bacterial extracts (4) were made from the original phage clone integrated by lysogeny. Extract from a bacteria lysogenized with another plaque (which did not bind to CRE sites) from the screen was used as a negative control. Competition experiments were done using a 4-100 fold molar excess (relative to the probe) of unlabeled, wild-type CRE oligonucleotides or unlabeled, mutant CRE oligonucleotides.

Northern Blots

Total head and body RNA was isolated from flies according to the protocol of (17). Total RNA from all other developmental stages was a gift from Eric Schaeffer. All RNA samples were selected twice on oligo-dT columns (5 Prime-3 Prime) to isolate poly A+ RNA. Two μg of poly A+ RNA was fractionated on 1.2% formaldehyde-formamide agarose gels, transferred to nitrocellulose and probed using an uniformly labeled, strand-specific, antisense RNA (aRNA) probe. The template for the synthesis of aRNA was one of the partial cDNA clones isolated from the library screen (pJY199). This cDNA contained the carboxyl-terminal 86 amino acids of the dCREB2-b protein and about 585 bp of 3' untranslated mRNA. All Northern blots were washed at high stringency (0.1% SDS, 0.1×SSC, 65° C.).

In situ Hybridization To Tissue Sections

Frozen frontal sections were cut and processed under RNAse-free conditions, essentially as described in (58), with modifications for riboprobes as noted here. Digoxigenin-labeled riboprobes were made from pJY199 using the Genius kit (Boehringer-Mannheim). One μg of Xba-linearized template and T3 RNA polymerase was used to make the antisense probe, while one μg of EcoRI-linearized template together with T7 RNA polymerase was used for the control sense probe. Alkaline hydrolysis (30 minutes at 60° C.) was used to reduce the average probe size to about 200 bases. The hydrolyzed probe was diluted 1:250 in hybridization solution (58), boiled, quickly cooled on ice, added to the slides and hybridized at 42° C. overnight. The slides were then treated with RNAse A (20 μg/ml RNAse A in 0.5M NaCl/10 mM Tris pH8 for 1 hour at 37° C.) prior to two 50° C. washes. Digoxigenin detection was as described.

Reverse Transcription Coupled With the Polymerase Chain Reaction (RT-PCR) Analysis of dCREB2 and Identification of Alternatively Spliced Exons

The template for reverse transcription coupled with the polymerase chain reaction (RT-PCR) was total RNA or poly A+ RNA isolated from Drosophila heads as in (17). Total RNA used was exhaustively digested with RNase-free DNase I (50 μg of RNA digested with 50 units of DNase I for 60-90' at 37° C. followed by phenol, phenol/chloroform extraction, and ethanol precipitation) prior to use. Results from separate experiments indicate that this DNase-treatment effectively eliminates the possibility of PCR products derived from any contaminating genomic DNA. Two rounds of selection using commercial oligo-dT columns (5 Prime-3 Prime) were used to isolate poly A+ RNA from total RNA. The template for an individual reaction was either 100-200 ng of total RNA, or 10-20 ng of poly A+ RNA.

The RT-PCR reactions were performed following the specifications of the supplier (Perkin-Elmer) with a "Hot Start" modification (Perkin Elmer RT-PCR kit instructions). All components of the RT reaction, except the rTth enzyme, were assembled at 75° C., and the reaction was initiated by adding the enzyme and lowering the temperature to 70° C. At the end of 15 minutes, the preheated (to 75° C.) PCR components (including trace amounts of [α³² P]dCTP) were added quickly, the reaction tubes were put into a pre-heated thermocycler, and the PCR amplification begun. Cycling parameters for reactions (100 μl total volume) in a Perkin-Elmer 480 thermocycler were 94° C. for 60 seconds, followed by 70° C. for 90 seconds. For reactions (50 μl) in an MJ Minicycler the parameters were 94° C. for 45 seconds and 70° C. for 90 seconds.

All primers used in these procedures were designed to have 26 nucleotides complementary to their target sequence. Some primers had additional nucleotides for restriction sites at their 5' ends to facilitate subsequent cloning of the products. Primers were designed to have about 50% GC content, with a G or C nucleotide at their 3' most end and with no G/C runs longer than 3. For RT-PCR reactions with a given pair of primers, the Mg⁺² concentration was optimized by running a series of pilot reactions, at Mg⁺² concentrations ranging from 0.6 mM to 3.0 mM. Reaction products were analyzed on denaturing urea-polyacrylamide gels by autoradiography. Any product that appeared larger than the band predicted from the cDNA sequence was purified from a preparative native gel, re-amplified using the same primers, gel-purified, subcloned and sequenced.

To verify that a given RT-PCR product was truly derived from RNA, control reactions were run to show that the appearance of the product was eliminated by RNase A treatment of the template RNA. Also, products generated from reactions using total RNA as the template were re-isolated from reactions using twice-selected polyA+RNA as template.

Plasmids

Expression constructs for transient transfection experiments in Drosophila were made in the expression vector pAct5CPPA (35) or in pAcQ. pAcQ is a close derivative of pAct5CPPA in which the XbaI site at the 5' end of the 2.5 kb actin promoter fragment was destroyed and additional sites were inserted in the polylinker. pAc-dCREB1 was made by subcloning a KpnI-SacI fragment containing the complete dCREB1 open reading frame (from a cDNA subcloned into pKS+) into pAct5CPPA. pAc-PKA was constructed by subcloning an EcoRV fragment encoding the Drosophila PKA catalytic subunit (23) from a modified pHSREM1 construct (17) into pAct5CPPA. To make the 3×CRE-lacZ reporter construct for Drosophila cell culture, the double-stranded, wild-type 3×CRE oligonucleotide used in the gel shift analysis was cloned into the KpnI-XbaI backbone of HZ50PL (37), a reporter construct made for enhancer testing which has cloning sites in front of a minimal hsp70 promoter-lacZ fusion gene.

RSV-dCREB.2-a was constructed in a long series of cloning steps. Details of the construction are available upon request. Essentially, the activator-encoding open reading frame was first reconstructed on the plasmid pKS+ by sequentially adding each of the three exons (exons 2, 4 and 6) into the original cDNA of dCREB2-b, which had been subcloned from phage DNA into pKS+. Site-directed mutagenesis was used to introduce unique restriction enzyme sites both 5' and 3' of the dCREB2-b open reading frame, and these sites facilitated the subcloning process and allowed removal of 5' and 3' untranslated sequences. Once the activator was assembled, the resulting open reading frame was sequenced to confirm the cloning steps and moved into a modified RSV vector which contained a polylinker located between the RSV promoter and the SV40 polyadenylation sequences (RSV-0). RSV-dCREB2-b was made by moving the original dCREB2-b cDNA (which had been subcloned into pKS+) into RSV-0.

Other constructs used in experiments were: pCaE (pMtC) (52), which contains the cDNA for mouse PKA catalytic subunit cloned under the mouse metallothionein 1 promoter; RSV-βgal (20), which expresses the lacZ gene under control of the Rous sarcoma long terminal repeat promoter (29); RSV-CREB (28) is a CREB cDNA fragment containing the 341-amino acid open reading frame under the RSV LTR-promoter in RSV-SG, and the D(-71) CAT reporter (56) which is a fusion of a CRE-containing fragment of the rat somatostatin promoter and the bacterial CAT coding region.

F9 Cell Culture and Transfection

Undifferentiated F9 cells were maintained and transfected using the calcium phosphate method as described in (12), except that chloroquine was added to 100 mM immediately before transfection and precipitates were washed off ten hours after transfection, at which time the dishes received fresh chloroquine-free medium. Amounts of DNA in transfections were made equivalent by adding RSV-0 where required. Cells were harvested 30 hours after transfection. Extracts were made by three cycles of freeze/thawing, with brief vortexing between cycles. Particulate matter was cleared from extracts by ten minutes of centrifugation in the cold. β-galactosidase assays were performed as described in (53). CAT assays were performed as described in (65) using aliquots of extract heat-treated at 65° C. for ten minutes and centrifuged for ten minutes to remove debris. Results reported are from three experiments run on different days with at least three dishes per condition within each experiment. Error bars represent standard error of the mean, with error propagation taken into account (30).

Drosophila Cell Culture and Transient Transfection

Schneider L2 cells in Schneider's medium (Sigma) supplemented with 10 μl fetal bovine serum (FBS) or Kc167 cells in D-22 medium (Sigma) supplemented with 10% FBS, were transfected by the calcium phosphate method essentially as described in (41), with the following differences. Kc167 cells were plated at 2×10⁶ cells/ml and chloroquine was added to a final concentration of 100 mM immediately prior to transfection. We used a total of 10 μg of plasmid DNA per dish for L2 transfections and 25 μg per dish for Kc167 transfections. DNA masses in transfections were made equivalent with pGEM7Zf+ where required. Precipitates were left undisturbed on L2 cells until harvest, but for Kc167 cells the original medium was replaced with fresh, chloroquine-free medium after twelve hours. Cells were harvested thirty-six to forty-eight hours after transfection. Extracts were made and enzymatic assays performed as described above for F9 cells. Results reported for transfections are averages of at least three experiments run on different days, with at least duplicate dishes for each condition within experiments. Error bars represent standard error of the mean, with error propagation taken into account (30).

β-galactosidase (βgal) and Chloramphenicol Acetyl Transferase (CAT) Assays

β-galactosidase assays were run and activity calculated as described in (53). CAT assays were performed essentially according to (65) using supernatants from heat-treated aliquots of extracts (65° C. for 10 minutes and then centrifuged for 10 minutes). Relative activity was calculated according to (65).

PKA-Responsive Transcriptional Activation by dCREB2-a

F9 cells were transiently transfected with 10 μg of D(-71) CAT plasmid as a CRE-directed reporter. 5 μg of RSV-βgal reporter was included in each dish as a normalization control for transfection efficiency. Different groups received 8 μg of dCREB2-a expression vector and 4 μg of PKA expression vector, separately or in combination. Results are expressed as CAT/βgal enzyme activity ratios, standardized to values obtained with PKA-transfected dishes.

Transcriptional Effect of dCREB2-b and a Mutant Variant On PKA-Responsive Activation by dCREB2-a

F9 cells were transiently cotransfected with 10 μg of D(-71) CAT along with the indicated combinations of the following expression constructs: RSV-dCREB2-a (5 μg); pMtC (2 μg); RSV-dCREB2-b (5 μg); and RSV-mLZ-dCREB2-b, which expresses a leucine-zipper mutant of dCREB2-b (5 μg). The DNA mass for each dish was made up to 27 μg with RSV-O. Other experimental conditions are as described above under "PKA-Responsive Transcriptional Activation by dCREB2-a".

Transcriptional Activation of a CRE Reporter Gene by dCREB1 in Drosophila Schneider L2 cell culture

The cells were transiently transfected with a dCREB1 expression construct (1 μg), with or without a construct which expresses Drosophila PKA. 3×CRE-βgal reporter (1 μg) and the normalization Ac-CAT reporter (1 μg) were included in each dish. Expression vectors not present in particular dishes were replaced by pACQ.

Example 1 Isolation and Characterization of dCREB2

Two different genes were isolated in a DNA-binding expression screen of a Drosophila head cDNA library using a probe containing three CRE sites (3×CRE). Many clones were obtained for the dCREB2 gene, while only one clone was obtained for dCREB1. The dCREB2 clones had two alternatively-spliced open reading frames, dCREB2-b and dCREB2-c (see FIG. 2). These differed only in the presence or absence of exon 4 and in their 5' and 3' untranslated regions. The inferred translation product of dCREB2-b showed very high sequence similarity to the amino acid sequences of the basic region/leucine zipper (bZIP) domains of mammalian CREB (SEQ ID NO.: 4), CREM (SEQ ID NO.: 5) and ATF-1 (SEQ ID NO.: 6) (see FIG. 1B).

Chromosomal in situ hybridization using a dCREB2 probe localized the gene to a diffuse band at 17A2 on the X chromosome, a region which contains several lethal complementation groups (19).

To determine the DNA binding properties of dCREB2-b, the DNA binding activity of dCREB2-b was assayed using a gel mobility-shift assay. Bacterial extracts expressing the dCREB2-b protein retarded the migration of a triplicated CRE probe (3×CRE). The protein had lower, but detectable, affinity for a mutated 3×CRE oligonucleotide. Competition experiments using unlabeled competitor oligonucleotides showed that the binding of dCREB2-b to 3×CRE was specific with higher affinity for CRE sites than to nonspecific DNA. Together with the conserved amino acid sequence, this functional similarity suggested that dCREB2 was a CREB family member.

The expression pattern of dCREB2 was determined by Northern blot analysis of poly A+RNA from various developmental stages. A complex pattern with at least 12 different transcript sizes was found. Two bands of approximately 0.8 and 3.5 kb were common to all of the stages. The adult head contained transcripts of at least six sizes (0.8, 1.2, 1.6, 1.9, 2.3 and 3.5 kb). In situ hybridization to RNA in Drosophila head tissue sections showed staining in all cells. In the brain, cell bodies but not neuropil were stained.

dCREB2 has alternatively-spliced forms. Initial transfection experiments showed that the dCREB2-c isoform was not a PKA-responsive transcriptional activator. This information, together with the complex developmental expression pattern and the known use of alternative splicing of the CREM gene to generate PKA-responsive activators (24, 26) suggested that additional domains might be required to code for an activator.

Reverse transcription coupled with the polymerase chain reaction (RT-PCR) was used to identify new exons. Comparison of the genomic DNA sequence with that of cDNAs indicated the general exon/intron organization and assisted in the search for additional exons. Sense and antisense primers spaced across the dCREB2-b cDNA were synthesized and used pairwise in RT-PCR reactions primed with Drosophila head RNA. Reactions with primers in exons 5 and 7 (see FIG. 2) generated two products, one with the predicted size (compared with the cDNA clones) and one that was larger. The larger fragment was cloned and its sequence suggested the presence of exon 6 (see FIG. 1A; SEQ ID NO.: 1). A primer within exon 6 was synthesized, end-labeled and used to screen a Drosophila head cDNA library. Two clones were isolated, sequenced and found to be identical. This splicing isoform, dCREB2-d, confirmed the splice junctions and exon sequence inferred from the RT-PCR products.

Initial attempts to isolate exon 2 proved difficult. The genomic sequence that separated exons 1 and 3 (see FIG. 2) was examined and one relatively extensive open reading frame (ORF) was identified. Three antisense primers, only one of which lay inside this ORF, were synthesized based on the intron sequence. Three sets of RT-PCR reactions were run in parallel, each using one of the three antisense primers and a sense primer in exon 1. Only the reaction that used the antisense primer in the ORF produced a PCR product. The sequence of this product matched a continuous stretch of nucleotides from the genomic sequence, extending 3' from exon 1 past the splice junction in the dCREB2-b cDNA to the location of the antisense primer in the ORF. This fragment suggested that exon 1 might be extended in some mRNAs by use of an alternative 5' splice site located 3' to the site used to make dCREB2-b. Based on the newly-identified exon sequences, a sense primer was made. This primer was used with an antisense primer in exon 3 to generate a new product whose sequence established the location of the new 5' splice site. The sequence added to exon 1 by alternative 5' splice site selection is denoted exon 2. The exon 2 sequence also showed that the same 3' splice site was used both for the original cDNA and for exon 2. To independently verify this alternative splicing pattern, we carried out RT-PCR with a primer that spanned the 3' splice junction of exon 2 and a primer in exon 1. The sequence of the product corroborated the splice junctions of exon 2 shown in FIG. 1A (SEQ ID NO.: 1).

To determine if exons 2 and 6 could be spliced into the same molecule, we carried out an RT-PCR reaction with primers in exons 2 and 6. The reaction produced a product of the size predicted by coordinate splicing of these two exons and the identity of this product was confirmed by extensive restriction analysis.

dCREB2 is a Drosophila CREB/ATF gene. FIG. 1A shows the DNA sequence (SEQ ID NO.: 1) and inferred amino acid sequence (SEQ ID NO.: 2) of dCREB2-a, the longest ORF that can result from the identified alternative splicing events. The indicated translation start site for this ORF is probably authentic because: i) stop codons occur upstream from this ATG in all reading frames in our dCREB2 cDNAs (sequences not shown) ii) this ATG was selected by computer (65) as the best ribosome binding site in the DNA sequence that contains the ORF; and iii) use of the next ATG in the open reading frame 480 nucleotides downstream would not produce an inferred product that is a PKA-dependent activator (see below). This information does not exclude the possibility that internal translation initiation sites may be used in this transcript, as happens in the CREM gene's S-CREM isoform (15).

The dCREB2-a open reading frame predicts a protein of 361 amino acids with a carboxyl-terminal bZIP domain (SEQ ID NO.: 3) highly homologous to those of mammalian CREB (SEQ ID NO.: 4) and CREM (SEQ ID NO.: 5) (see FIG. 1B). The inferred dCREB2-a product has a small region of amino acids containing consensus phosphorylation sites (59) for PKA, calcium/calmodulin-dependent kinase II (CaM kinase II) and protein kinase C (PKC) at a position similar to that of the P-box in CREB, CREM and ATF-1. The amino-terminal third of the predicted dCREB2-a is rich in glutamines (including runs of four and five residues). Glutamine-rich activation domains occur in CREB, CREM, and other eukaryotic transcription factors, including some from Drosophila (11,54).

A computerized amino acid sequence homology search (71) with the predicted dCREB2-a protein sequence (SEQ ID NO.: 2) identifies CREB, CREM and ATF-1 gene products as the closest matches to dCREB2-a. The homology is particularly striking in the carboxyl-terminal bZIP domain where 49 of 54 amino acids are identical with their counterparts in mammalian CREB (FIG. 1B). The homology is less striking, albeit substantial, in the activation domain. Lower conservation in this domain is also characteristic of the mammalian CREB and CREM genes (48).

FIG. 2 shows the exon organization of all of the dCREB2 alternative splice forms that we have detected, both as cDNAs and by RT-PCR. Splice products of dCREB2 fall into two broad categories. One class of transcripts (dCREB2-a, -b, -c, -d) employs alternative splicing of exons 2, 4 and 6 to produce isoforms whose protein products all have the bZIP domains attached to different versions of the activation domain. The second class of transcripts (dCREB2-q, -r, -s) all have splice sites which result in in-frame stop codons at various positions upstream of the bZIP domain. These all predict truncated activation domains without dimeriation or DNA binding activity.

Two different dCREB2 isoforms, dCREB2-a and dCREB2-b, have opposite roles in PKA-responsive transcription. The capacity of isoforms of the dCREB2 gene to mediate PKA-responsive transcription was tested in F9 cells. These cells have been used extensively to study CREB-dependent activation because their endogenous cAMP-responsive transcription system is inactive (28, 49, 50). Candidate cAMP-responsive transcription factors, synthesized from expression vectors, were transiently transfected with and without a construct expressing the PKA catalytic subunit. CREB-dependent changes in gene expression were measured using a cotransfected construct that has a CRE-containing promoter fused to a reporter gene.

The product of the dCREB2-a isoform is a PKA-dependent activator of transcription (FIG. 3). Transfection of PKA or dCREB2-a alone gave only modest activation above baseline values. Cotransfection of dCREB2-a and PKA together, however, gave levels of activation 5.4-fold greater than the activation seen with PKA alone.

dCREB2-b did not act as a PKA-dependent transactivator. When transfected together with the reporter and PKA, it failed to stimulate reporter activity. Instead, it functioned as a direct antagonist of PKA-dependent activation by dCREB2-a (FIG. 4). Cotranfection of equimolar amounts of the dCREB2-a and dCREB2-b expression constructs, along with PKA and the reporter, resulted in a nearly complete block of PKA-dependent activation from the CRE-containing reporter.

The strong homology between the leucine zippers of dCREB2 (SEQ ID NO.: 3), CREB (SEQ ID NO.: 4) and CREM (SEQ ID NO.: 5) (see FIG. 1B) suggested that mutations which abolish CREB dimerization (18) should also affect dCREB2 dimerization. The mutant Drosophila molecule mLZ-dCREB2-b was made by introducing two single-base changes that convert the middle two leucines of the leucine zipper to valines. An identical mutation in CREB abolishes homodimerization in vitro (18). Cotransfected mLZ-dCREB2-b failed to block PKA-dependent activation by dCREB2-a (FIG. 4).

Example 2 Isolation and Characterization of dCREB1

A single cDNA representing the dCREB1 gene was isolated in the same screen of a Drosophila lambda gt11 expression library that yielded the dCREB2 cDNAS. The sequence of the dCREB1 cDNA contained a complete open reading frame specifying a 266 amino acid protein with a carboxyl-terminal leucine zipper four repeats long and an adjacent basic region (FIG. 5; SEQ ID NO.: 7). The amino-terminal half of the inferred protein contains an acid-rich activation domain, with glutamate, asparate and proline residues spaced throughout. dCREB1 has consensus phosphorylation sites for CaM kinase II and PKC throughout its length, but has no predicted phosphoacceptor site for PKA.

Gel shift analysis showed higher-affinity binding of the dCREB1 protein to 3×CRE than to 3×mCRE. Transcriptional activation by dCREB1 was assayed with transient cotransfection experiments using the Drosophila L2 and Kc167 cell culture lines. In L2 cells, dCREB1 activates transcription from CREs, but this effect is not enhanced by cotransfection of PKA (FIG. 6). In Kc167 cells, dCREB1 fails to activate reporter expression either alone or with cotransfected PKA expression constructs.

Genomic Southern blot analysis indicates that dCREB1 is a single copy gene, and chromosomal in situ hybridization shows that it is located at 54A on the right arm of chromosome 2.

These results show that dCREB1 is a non-PKA responsive CREB family member from Drosophila.

The following materials and methods were used in the work described in Examples 3 and 4.

Isolating Transgenic Flies

EcoRI restriction sites were added (using PCR) just 5' to the putative translation initiation site and just 3' to the translation termination site in the dCREB2-b cDNA. This fragment was sequenced and subcloned into CaSpeR hs43, a mini-white transformation vector which contains the hsp70 promoter, in the orientation so that the dCREB2-b open reading frame is regulated by the hsp70 promoter. Germ-line transformation was accomplished using standard techniques (Spradling and Rubin, 1982; Rubin and Spradling,1982). Two transgenic lines, 17-2 and M11-1, each with one independent P-element insertion were generated and characterized. They appeared normal in general appearance, fertility and viability. These transgenic lines were outcrossed for at least five generations to w(CS-10) (Dura et al., 1993), which itself had been outcrossed for ten generations to a wild-type (Can-S) stock. This extensive series of outcrossing is necessary to equilibrate the genetic background to that of Canton-S. Flies homozygous for the 17-2 transgene were bred and used for all experiments.

The mutant blocker has been described previously (see Example 1). The mutations were substituted into an otherwise wild-type blocker construct and germ-line transformants were made by injecting into w(isoCJ1) embryoes. Flies homozygous for the A2-2 transgene insertion were bred and used for all experiments. w(isoCJ1) is a subline of w(CS10) (see above) carrying isogenic X, 2^(nd) and 3^(rd) chromosomes and was constructed by Dr. C. Jones in our laboratory. Originally 40 such sublines were w(CS10) using standard chromosome balancer stocks. Olfactory acuity, shock reactivity, learning and three-hr memory after one-cycle training then were assayed in each isogenic subline. As expected, a range of scores among the sublines was obtained. w(isoCJ1) yielded scores that were most like those of w(CS10) on each of these assays. By injecting DNA into the relatively homogeneous genetic background of w(isoCJ1), outcrossing of the resulting germ-line transformants to equilibrate heterogeneous) genetic backgrounds was not necessary.

Cycloheximide Feeding and Heat-Shock Regimen

Flies were fed cycloheximide as reported (Tully et al., 1994) except that the feeding period was limited to 12-14 hrs prior to training, or to the 24-hr retention interval after training. Flies which were fed prior to training were transferred directly to the training apparatus after feeding, subjected to massed or spaced training, then transferred to test tubes containing filter paper strips soaked with 5% glucose during the 24-hr interval. Flies which were fed after training were trained, then transferred immediately to test tubes containing filter paper strips soaked with 5% glucose solution which was laced with 35 mM CXM. Flies remained in the test tubes for the duration of the 24-hr retention interval.

For heat-shock induction, flies were collected within two days of eclosion, placed in glass bottles in groups of about 600, and incubated overnight at 25° C. and 70% relative humidity. The next day, three hours before training, groups of approximately 100 flies were transferred to foam-stoppered glass shell vials containing a strip of filter paper to absorb excess moisture. The vials then were submerged in a 37° C. water bath until the bottom of the foam stopper (inside the vial) was below the surface of the water, thereby insuring that the flies could not escape heat-shock. The vial remained submerged for 30 min, after which the flies were transferred to a standard food vial for a 3-hr recovery period at 25° C. and 70% relative humidity. Training began immediately after the recovery period.

Pavlovian Learning and Memory and Testing

Flies were trained with an automated version of the learning procedure of Tully and Quinn (1985; Tully et al., 1994). Groups of about 100 flies were exposed sequentially to two odors [either octanol (OCT) or methylcyclohexanol (MCH)] for 60 seconds with 45 seconds rest intervals after each odor presenation. During exposure to the first odor, flies also were subjected to twelve 1.5-second pulses of 60 V DC with a 5-second interpulse interval. After training, flies were transferred to food vials for a particular retention interval. Conditioned odor-avoidance responses then were tested by transferring flies to the choice point of a T-maze, where they were exposed simultaneously to OCT and MCH carried in the distal ends of the T-maze arms and out the choice point on converging currents of air. Flies were allowed to distribute themselves in the T-maze arms for two minutes, after which they were trapped in their respective arms, anesthetized and counted. The "percent correct" then was calculated as the number of flies avoiding the shock-paired odor (they were in the opposite T-maze arm) divided by the total number of flies in both arms. (The number of flies left at the choice point, which usually was less than 5%, were not included in this calculation). Finally, a performance index (PI) was calculated by averaging the percent corrects of two reciprocal groups of flies--one where OCT and shock were paired, the other where MCH and shock were paired--and then by normalizing the average so that a PI=0 represented a 50:50 distribution in the T-maze and a PI=100 represented 100% avoidance of the shock-paired odor. For these studies, three different training protocols were used: 1. One-cycle training consisted of the training session just described. 2. Massed training consisted of 10 of these training cycles delivered one right after the other. 3. Spaced training consisted of 10 training cycles with a 15-min rest interval between each. One-cycle training was used to assay learning, while massed and spaced was used to assay consolidated memories (Tully et al., 1994).

Olfactory Acuity and Shock Reactivity

Odor avoidance responses to OCT or to MCH at two different concentrations--one (10⁰) usually used in conditioning experiments and a 100-fold (10⁻²) dilution thereof--were quantified in various groups of flies in the absence of heat shock and 3 hr or 24 hr after heat shock with the method of Boynton and Tully (1992). Briefly, flies are placed in a T-maze and given a choice between an odor and air. The odors are naturally aversive, and flies ususally choose air and avoid the T-maze arm containing the odor. For shock reactivity, flies are given a choice between an electrified grid in one T-maze arm, and an unconnected grid in the other. After the flies have distributed themselves, they are anesthetized, counted and a PI is calculated.

Statistical Analyses of Behavioral Data

Since each PI is an average of two percentages, the Central Limit Theorem predicts that they should be distributed normally (see Sokal and Rohlf, 1981). This expectation was shown to be true by an empirical determination with data from Tully and Quinn (1985) and Tully and Gold (1993). Thus, untransformed (raw) data were analyzed parametrically with JMP2.1 statistical software (SAS Institute Inc., Cary N.C.). Since preliminary experiments preceded all of the experiments summarized herein, all pairwise comparisons were planned. To maintain an experimentwise error rate of alpha=0.05, the critical P values for these individual comparisons were adjusted accordingly (Sokal and Rohlf, 1981) and are listed below for each experiment.

All experiments were designed in a balanced fashion with N=2 PIs per group collected per day; then replicated days were added to generate final Ns. In each experiment, the experimenter (M.D.) was blind to genotype.

A. One-day memory in wild-type flies fed CXM before or immediately after massed or spaced training (FIG. 8): PIs from these four drug treatments (-CXM before, -CXM after, +CXM before and +CXM after) and two training procedures (massed and spaced) were subjected to a TWO-WAY ANOVA with DRUG (F.sub.(3,56) =8.93; P<0.001) and TRAINing (F.sub.(1,56) =18.10, P<0.001) as main effects and DRUG×TRAIN (F.sub.(3,56) =4.68, P=0.006) as the interaction term. P values from subsequent planned comparisons are summarized in FIG. 8. The six planned comparisons were judged significant if P≦0.01.

B. One-day memory after massed or spaced training in dCREB2-b transgenic flies (FIGS. 9A and 9B): In experiments with the 17-2 transgenic line, PIs from two strains (Can-S and 17-2) and four training-regimens (spaced-hs, spaced+hs, massed-hs and massed+hs) were subjected to a TWO-WAY ANOVA with STRAIN (F.sub.(1,40) =1.57; P=0.22) and TRAINing-regimen (F.sub.(3,40) =25.81, P<0.001) as main effects and STRAIN×TRAIN (F.sub.(3,40) =6.62, P=0.001) as the interaction term. A similar analysis was done with data from the M11-1 transgenic line, yielding STRAIN (F.sub.(1,40) =2.81; P=0.10), TRAINing-regimen (F.sub.(3,40) =11.97, P<0.001) and STRAIN×TRAIN (F.sub.(3,40) =3.37, P=0.03) effects. P values from subsequent planned comparisons are summarized in FIGS. 9A and 9B. In each experiment, the seven planned comparisons were judged significant if P≦5 0.01.

C. Learning after one-cycle training in 17-2 transgenic flies (FIG. 9C): PIs from two strains (Can-S and 17-2) and three heat-shock regimens [-hs, +hs (3 hr) and+hs (24 hr)] were subjected to a TWO-WAY ANOVA with STRAIN (F.sub.(1,30) =0.69; P=0.41) and HEAT-shock regimen (F.sub.(2,30) =10.29, P<0.001) as main effects and STRAIN×HEAT (F.sub.(2,30) =0.71, P=0.50) as the interaction term. P values from subsequent planned comparisons are summarized in FIG. 9C. The three planned comparisons were judged significant if P≦0.02.

D. One-day memory after spaced training in A2-2 transgenic flies (FIG. 10): PIs from these three strains [w(isoCJ1), 17-2 and A2-2] and two heat-shock regimens [-hs and +hs (3 hr)] were subjected to a TWO-WAY ANOVA with STRAIN (F.sub.(2,30) =9.43, P<0.001) and HEAT-shock regimen (F.sub.(1,30) =9.84, P=0.004) as main effects and STRAIN×HEAT (F.sub.(2,30) =5.71, P=0.008) as the interaction term. P values from subsequent comparisons are summarized in FIG. 10. The six planned comparisons were judged significant if P≦0.01.

E. Olfactory acuity in 17-2 flies (Table): PIs from these two strains (Can-S and 17-2), four different odor-levels (OCT- 10⁰, OCT- 10⁻², MCH-10⁰ and MCH-10⁻²) and three heat-shock regimens [-hs. +hs (3 hr) and +hs (24 hr)] were subjected to a THREE-WAY ANOVA with STRAIN (F.sub.(1,184) =0.12, P=0.73), ODOR-level (F.sub.(3,184) =126.77, P<0.001) and HEAT-shock regimen (F.sub.(2,184) =3.55, P=0.03) as main effects, STRAIN×ODOR (F.sub.(3,184) =1.23, P=1.23, P=0.30), STRAIN×HEAT (F.sub.(2,184) =0.33, P=0.72) and ODOR×HEAT (F.sub.(6,184) =3.14, P=0.006) as two-way interaction terms and STRAIN×ODOR×HEAT (F.sub.(6,184) =0.48, P=0.83) as the three-way interaction term. P values from subsequent planned comparisons are summarized in the Table. The twelve planned comparisons were significant if P≦0.005.

F. Shock reactivity in 17-2 flies (Table): PIs from these two strains (Can-S and 17-2), two shock groups (60 V and 20 V) and three heat-shock regimens [-hs, +hs (3 hr) and +hs (24 hr)] were subjected to a THREE-WAY ANOVA with STRAIN (F.sub.(1,84) =0.50, P=0.48), SHOCK (F.sub.(1,84) =97.78, P<0.001) and HEAT-shock regimen (F.sub.(2,84) =3.36, P=0.04) as main effects, STRAIN×SHOCK (F.sub.(1,84) =1.12, p=0.29), STRAIN×HEAT (F.sub.(2,84) =1.06, P=0.35) and SHOCK×HEAT (F.sub.(2,84) =6.66, P=0.002) as two-way interaction terms and STRAIN×SHOCK×HEAT (F.sub.(2,84) =1.75, P=0.18) as the three-way interaction term. P values from subsequent planned comparisons are summarized in the Table. The six planned comparisons were judged significant if P≦-0.01.

G. Seven-day memory after spaced training in 17-2 flies (FIG. 11): PIs from two strains (Can-S and 17-2) and two heat-shock regimens [-hs and +hs(3 hr)] were subjected to a TWO-WAY ANOVA with STRAIN (F.sub.(1,20) =6.09; P=0.02) and HEAT-shock regimen (F.sub.(1,20) =16.30, P=0.001) as main effects and STRAIN×TRAIN (F.sub.(1,20) =7.73, P=0.01) as the interaction term. P values from subsequent planned comparisons are summarized in FIG. 11. The three planned comparisons were judged significant if P≦0.02.

H. One-day memory after spaced training in rsh;17-2 double mutants (FIG. 12): PIs from three strains (17-2, rsh and rsh;17-2) and two heat-shock regimens [-hs and +hs (3 hr)] were subjected to a TWO WAY ANOVA with STRAIN (F.sub.(2,30) =32.05; P<)0.001) and HEAT-shock regimen (F.sub.(1,30) =59.68, P<0.001) as main effects and STRAIN×TRAIN (F.sub.(2,30) =11.59, P<0.001) as the interaction term. P values from subsequent planned comparisons are summarized in FIG. 12. The five planned comparisons were judged significant if P≦0.01.

I. Learning after one-cycle training in rsh;17-2 mutants (see text): PIs from these two strains (Can-S and rsh;17-2) and two heat-shock regimens [-hs and +hs (3 hr)] were subjected to a TWO-WAY ANOVA with STRAIN (F.sub.(1,20) =86.85, P<0.001) and HEAT-shock regimen (F.sub.(1,20) =0.02, P<0.89) as main effects and STRAIN×HEAT (F.sub.(1,20) =0.86, P=0.37) as the interaction term. P values from subsequent planned comparisons are summarized in the Table. The two planned comparisons were significant if P≦0.03.

J. Olfactory acuity in rsh;17-2 flies (Table): PIs from these two strains (Can-S and rsh;17-2), four different odor-levels (OCT-10⁰, OCT-10⁻², MCH-10⁰ and MCH-10⁻²) and two heat-shock regimens [-hs, and +hs (3 hr)] were subjected to a THREE-WAY ANOVA with STRAIN (F.sub.(1,112) =0.02, P=0.88), ODOR-level (F.sub.(3,112) =50.03, P<0.001) and HEAT-shock regimen (F.sub.(1,112) =29.86, P<0.001) as main effects, STRAIN×ODOR (F₃,112) =2.15, P=0.10), STRAIN×HEAT (F.sub.(1,112) =0.34, P=0.56) and ODOR×HEAT (F.sub.(3,112) =6.41, P=0.001) as two-way interaction terms and STRAIN×ODOR×HEAT (F.sub.(3,112) =1.12, P=0.35) as the three-way interaction term. P values from subsequent planned comparisons are summarized in the Table. The eight planned comparisons were judged significant if P≦0.01.

K. Shock reactivity in rsh;17-2 flies (Table): PIs from these two strains (Can-S and rsh;17-2), two shock groups (60 V and 20 V) and two heat-shock regimens [-hs and +hs (3 hr)] were subjected to THREE-WAY ANOVA with STRAIN (F.sub.(1,56) =0.51, P=0.48), SHOCK (F.sub.(1,56) =88.14, P<0.001) and HEAT-shock regimen (F.sub.(1,56) =0.08, P=0.77) as main effects, STRAIN×SHOCK (F₁,56) =0.12, P=0.73, STRAIN×HEAT (F.sub.(1,56) =0.03, P=0.86) and SHOCK×HEAT (F.sub.(1,56) =0.39, P=0.53) as two-way interaction terms and STRAIN×SHOCK×HEAT (F.sub.(1,84) =1.58, P=0.21) as the three-way interaction term. P values from subsequent planned comparisons are summarized in the Table. The four planned comparisons were judged significant if P≦0.01.

Northern Analysis

For RNA collection, the heat-shock regimen was the same as for behavioral experiments. For any indicated time interval between heat-shock and collection, flies rested in food-containing vials at 25° C. Flies were collected and quickly frozen in liquid nitrogen. All Northern analyses used head RNA. The tube of frozen flies was repeatedly rapped sharply on a hard surface, causing the heads to fall off. The detached frozen heads were recovered by sieving on dry ice. Approximately 1000 heads were pooled for RNA preparation. Wild-type and transgenic flies for each individual time point always were processed in parallel. Flies that were not induced were handled in a similar manner to induced flies, except that the vials were not placed at 37° C. Total head RNA was isolated from each group of flies, and poly A+ RNA was isolated using oligo dT columns according to the instructions of the manufacturer (5'→3' Inc.). The concentration of poly A+ mRNA was measured spectrophotometrically, and 0.5 mg of mRNA per lane was loaded and run on 1.2% formaldehyde-agarose gels. Northern blots were prepared, probed and washed (0.1×SSC at 65° C.) as described (Ausubel et al., 1994). For detection of the transgene, an 843 bp dCREB2-b cDNA fragment was subcloned into pKS+ and used to generate a uniformly-labeled antisense riboprobe. This fragment codes for the carboxyl-terminal 86 amino acids of the dCREB2-b protein plus 3' untranslated mRNA.

Western Blot Analysis and Antiserum

Western blot analysis was performed using a rabbit antiserum raised against a peptide corresponding to 16 amino acids in the basic region of the dCREB2-b cDNA with an additional COOH terminal Cys. The sequence of the peptide was: (SEQ ID NO.: 24) NH2-RKREIRLQKNREAAREC-COOH. The peptide was synthesized and coupled to Sulfo-SMCC (Pierce) activated keyhole lympet hemocyanin. The antigen was injected into rabbits (100 ug) and boosted at two week intervals. Sera was bled and tested for immune reactivity towards bacterially-expressed dCREB2-b protein. The antiserum was passed through a CM Affi-gel Blue column (Biorad), and the flow-through was concentrated by ammonium sulfate precipitation, resuspended and dialyzed against PBS (0.14 M NaCl, 2.7 mM KCl, 4.3 mM Na₂ HPO₄ 7H₂ O, 1.4 mM KH₂ PO₄, pH 7.3). The dialyzed serum was affinity-purified using a peptide column made using an Ag/Ab Immobilization kit (Immunopure from Pierce). After the antiserum was eluted using a 4M MgCl₂, 0.1 M HEPES pH 6.0 buffer, it was dialyzed into PBS and frozen.

Each data point represents approximately 5 fly heads. Groups of about 25-50 flies were collected and quickly frozen on liquid nitrogen until all of the time points had been collected. Heads were isolated resuspended in approximately 200 ul of 1× Laemmli sample buffer, allowed to thaw and homogenized with a Dounce type B pestle. Samples were boiled for 5 minutes, and centrifuged for 10 minutes at room temperature in an Eppendoff microcentrifuge. The supernatants were collected and boiled again just prior to loading onto protein gels. Standard procedures were used to separate equal amounts of proteins from each sample on 12% polyacrylamide-SDS gels and to transfer them to PVDF membranes by electroblotting (Ausubel et al., 1994).

The membranes were blocked for 60 minutes with a 5% BSA solution made up in TBST (10 mM Tris 50 mM NaCl, 0.05% Tween 20). The primary antibody was diluted 1:1000 in TBST and incubated with the filter for 30 minutes. The membranes were washed three times with TBST for 5 minutes each time and then incubated for 30 minutes with an alkaline phosphatase-conjugated anti-rabbit IgG second antibody (Promega) diluted 1:7500 in TBST. The membranes were washed three more times as before and developed using a chromogenic alkaline phosphatase reaction according to manufacturers suggestions (Promega).

Example 3 Transgene Expression Increased After Heat-Shock Induction

In order to interpret the effects of transgene induction on behavior, dCREB2-b expression in transgenic flies (17-2) after heat-shock induction was measured. Northern blot analysis revealed elevated levels of hs-dCREB2-b message in the 17-2 flies immediately and three hours after heat-shock (FIG. 7A). This induction was also detectable in brain cells using in situ hybridization. Western blot showed increased dCREB2-b protein immediately after induction (FIG. 7B). Elevated levels of the dCREB2-b protein were seen nine hours later and were still detectable twenty four hours after induction. These data indicate that increased amounts of dCREB2-b existed in brain cells throughout spaced training, which ended about six hours after heat induction.

The behavioral experiments also used transgenic flies (A2-2) which expressed a mutated dCREB2-b protein (dCREB2-mLZ). These mutations changed the two internal leucine residues of the leucine zipper to valine residues, and these changes have been shown to result in a protein which is unable to form dimers (Dwarki et al., 1990). In transient co-transfection assays, the mutant protein was unable to block PKA-dependent transcription mediated by dCREB2-a, while the wild-type protein had blocking function. Western blot analysis showed that the wild-type and mutant blocker are expressed at similar levels beginning immediately after heat-shock induction and lasting for at least 6 hours (FIG. 7C). Therefore, it is unlikely that these two proteins have large differences in expression levels or stability in the transgenic flies.

Northern blot analysis of two different housekeeping genes, myosin light chain (Parker et al., 1985) and elongation factor a (Hovemann et al., 1988), showed that steady-state levels of their RNAs were unaffected after transgene induction for at least 3 hours. Gel shift analysis using two different consensus DNA binding sites showed that there was no large effect on the gel shift species which formed after transgene induction for at least 9 hours. Cotransfection of the blocker did not interfere with the activity of a transcription factor from a different family in cell culture. Considered together, hs-dCREB2-b probably had fairly specific molecular modes of action after induction.

Example 4 Assessment of the Role of CREBs in Long-Term Memory Formation

To confirm and extend the results of Tully et al. (1994), flies were fed 35 mM cycloheximide (CXM) for 12-14 hours before, or for the 24-hr retention interval immediately after, massed or spaced training (FIG. 8). Each of these CXM feeding regimens significantly reduced one-day memory after spaced training but had no effect on one-day memory after massed training (FIG. 8). Thus, cyclohexmide feeding immediately before or after spaced training disrupts one-day memory. These results suggest that protein synthesis is required soon after training for the formation of long-lasting memory.

The results in FIG. 8 show that cycloheximide feeding affects one-day retention after spaced training but not massed training. Different groups of wild-type (Can-S) flies were fed 5% glucose solution alone (hatched bars) or laced with 35 mM CXM (striped bars) either for 12-14 hr overnight before massed or spaced training or for the 24-hr retention interval immediately after training. One-day memory retention was significantly lower than normal in flies fed CXM before (P<0.001) or after (P<0.001) spaced training. In both cases, one-day retention in CXM-fed flies was reduced to a level similar to one-day memory after massed training in glucose-fed flies (P=0.88 for CXM before training and P=0.71 for CXM after training). In contrast, no difference was detected between CXM-fed and control flies for one-day memory after massed training (P=0.49 and P=0.46, respectively).

One day retention after spaced training was unaffected in uninduced (-hs) transgenic flies (17-2) but was significantly reduced in induced (+hs) transgenic flies (FIG. 9A). In contrast, one-day retention after massed training was normal in both uninduced and induced transgenic flies (FIG. 9A). Comparisons of one-day retention after spaced or massed training between wild-type flies with (+hs) or without (-hs) heat-shock indicated that the heat-shock regimen itself did not have a non-specific effect on memory after either training protocol. Thus induction of the dCREB2-b transgene only affected (i.e., disrupted) one-day memory after spaced training.

One day retention after spaced or massed training in M11-1, a second line carrying an independent hs-dCREB2-b insertion, also was tested. Results with M11-1 were similar to those obtained with 17-2 (FIG. 9B). These results show that the effect of induced hs-dCREB2-b does not depend on any particular insertion site of the transgene.

The results in FIGS. 9A-9C show that induction of the dCREB2-b transgene disrupts one-day memory after spaced training, while one-day memory after massed training and learning are normal.

In FIG. 9A, different groups of wild-type (Can-S) flies (hatched bars) or hs-dCREB2-b transgenic (17-2) flies (striped bars) were given spaced training or massed training in the absence of heat shock (-hs) or three hours after heat shock (+hs). After training, flies were transferred to standard food vials and stored at 18° C. until one-day memory was assayed. No differences in one-day memory after spaced or massed training were detected between Can-S vs. 17-2 flies in the absence of heat shock (-hs; P=0.83 and 0.63, respectively). When flies were trained three hours after heat shock (+hs), however, one-day memory was significantly different between Can-S v. 17-2 flies after spaced training (P<0.001) but not after massed training (P=0.23). In fact, the one-day memory after spaced training was no different than that after massed training in induced 17-2 flies (P=0.59). In addition, the heat-shock regimen did not produce a non-specific effect on one-day retention after spaced (P=0.59) or massed (P=1.00) training in Can-S flies. N=6 performance indices (PIs) per group.

The experiment described in FIG. 9A was repeated in FIG. 9B with a second, independently derived dCREB2-b transgenic line, M11-1 (striped bars). Here again, a) no differences in one-day memory after spaced or massed training were detected between Can-S vs. M11-1 flies in the absence of heat-shock (-hs; P=0.83 and 0.86, respectively), b) a significant difference between Can-S v. M11-1 for one-day memory after spaced training (P<0.001) but not after massed training (P=0.85) when trained three hours after heat-shock (+hs), c) one-day memory after spaced training was no different than that after massed training in induced M11-1 flies (P=0.43) and d) the heat-shock regimen did not produce a non-specific effect on one-day retention after spaced (P=0.59) or massed (P=0.94) training in Can-S flies. N=6 PIs per group.

If induction of the transgene specifically affected LTM via disruption of gene expression, then learning should not be affected, since it does not require new protein synthesis (see Tully et al. 1994). Different groups of flies were trained using one-cycle training either without heat-shock, or three or twenty four hours after heat-shock. These time points after induction were selected to correspond to the times when flies were trained and tested in the previous experiments (see FIGS. 9A and 9B). Induction of the transgene (d-CREB2-b) in the 17-2 line had no effect on learning in either case (FIG. 9C).

In FIG. 9C, different groups of Can-S flies (hatched bars) or 17-2 transgenic flies (striped bars) received one-cycle training in the absence of heat shock (-hs) or three (+hs 3hr) or 24 (+hs 24hr) hours after heat-shock and then were tested immediately afterwards. In each case, no differences between Can-S vs. 17-2 flies were detected (Ps=0.28, 0.64 and 0.42, respectively), indicating that learning was normal in induced or uninduced transgenic flies. N=6 PIs per group.

Induction of the transgene which contained the mutant blocker (A2-2) did not affect one-day retention after spaced training, while the wild-type blocker (17-2) had a dramatic effect (FIG. 10). The w(iso CJ1) flies, whose one-day retention also was unaffected by heat induction, is the isogenic control for the mutant blocker transgenic flies. Since Western blot analysis showed that wild-type and mutant blockers probably have similar expression levels, this result suggests that the blocker requires an intact leucine zipper to function effectively.

FIG. 10 shows that induction of the hs-dCREB2-mLZ mutant blocker does not affect one-day retention after spaced training. Different groups of wild-type [w (iso CJ1)], hs-dCREB2-b transgenic (17-2) or mutant hs-dCREB2-mLZ transgenic flies (A2-2) received spaced training in the absence of heat-shock (-hs) or three hours after heat-shock (+hs). The flies were then handled and tested as in FIG. 9A. No differences in one-day memory after spaced training were detected between w(isoCJ1) vs. 17-2 flies or between w(isoCJ1) vs. A2-2 flies in the absence of heat shock (-hs; P=0.38 and 0.59, respectively). When flies were trained three hours after heat shock (+hs), however, one-day memory after spaced training was significantly different between w(isoCJ1) vs. 17-2 flies (P<0.001)--as in FIG. 9A--but was not different between w(isoCJ1) vs. A2-2 flies (P=0.78). In addition, the heat-shock regimen did not produce a non-specific effect on one-day retention after spaced training in w(isoCJ1) or A2-2 flies (P=0.40 and P=0.97, respectively. N=6 performance indices (PIs) per group.

Olfactory acuity and shock reactivity are component behaviors essential for flies to properly learn odor-shock associations. The Table shows the scores for these peripheral behaviors for Can-S versus 17-2 flies. With or without heat-shock, olfactory acuity and shock reactivity were normal in 17-2 transgenic flies. Thus, hs-dCREB2-b induction does not affect olfactory acuity or shock reactivity.

If induction of hs-dCREB2-b blocks long-term memory (LTM), then long-lasting memory also should be blocked. In wild-type flies, seven-day retention after spaced training consists solely of the CXM-sensitive LTM because the CXM insensitive ARM component has decayed away (Tully et al., 1994). In uninduced transgenic flies (17-2), seven-day retention after spaced training was similar to retention in uninduced wild-type flies (P=0.83; FIG. 11). Seven-day retention was severely disrupted, however, in transgenic flies which were trained three hours after heat-shock (P=0.001) and did not differ from zero (P=0.91). In contrast, the heat-shock protocol had no detectable effect on seven-day memory in wild-type flies (P=0.39). Thus, induction of hs-dCREB2-b disrupts long-term memory (LTM).

FIG. 11 shows that induction of hs-dCREB2-b completely abolishes 7-day memory retention. Previous analyses of radish mutants indicated that memory retention four or more days after spaced training reflects the sole presence of LTM. Thus, the effect of induced hs-dCREB2-b on LTM was verified by comparing 7-day retention after spaced training in Can-S (hatched bars) vs. 17-2 transgenic (striped bars) flies that were trained in the absence of heat-shock (-hs) or three hours after heat shock (+hs). Flies were stored in standard food vials at 18° C. during the retention interval. N=6 PIs per group. Seven-day retention after spaced training did not differ between Can-S and 17-2 in the absence of heat-shock (P=0.83) but was significantly lower than normal in 17-2 flies after heat-shock (P=0.002). In fact, 7-day retention after spaced training in induced 17-2 transgenic files was not different from zero (P=0.92). In addition, the heat-shock regimen did not affect 7-day retention after spaced training non-specifically in Can-S flies (P=0.39).

If induction of the hs-dCREB2-b transgene specifically blocks LTM, then it should only affect the CXM-sensitive component of consolidated memory after spaced training. For both transgenic lines, 17-2 and M11-1, the effect of transgene induction looked similar to the effect that CXM had on wild-type flies (compare FIG. 8 with FIGS. 9A and 9B). This similarity suggested that the induced dCREB2-b protein completely blocked CXM-sensitive memory, leaving ARM intact. The radish mutation disrupts ARM (Folkers et al., 1993), leaving only LTM one day after spaced training (Tully et al., 1994). Thus, a radish hs-dCREB2-b "double mutant" (rsh; 17-2) was constructed to allow examination of LTM in the absence of ARM. In the absence of heat-shock, rsh;17-2 double-mutants and radish single-gene mutants yielded equivalent one-day retention after spaced training (FIG. 12). In contrast, when these flies were heat-shocked three hours before spaced training, one-day retention was undetectable in rsh;17-2 flies but remained at mutant levels in radish flies. The double mutant also showed normal (radish-like) learning (P=0.59) and normal (wild-type) olfactory acuity and shock reactivity in the absence of heat-shock versus three hours after heat shock (see the Table).

                                      TABLE 1                                      __________________________________________________________________________     Olfactory acuity and shock reactivity in Can-S (wild-type), 17-2                 (hs-dCREB2-b transgenic) and rsh; 17-2 (radish, hs-dCREB2-b "double           mutant") flies.sup.a.                                                                   Olfactory Acuity  Shock                                              Heat      OCT      MCH      Reactivity                                         Shock                                                                              Group 10.sup.0*                                                                          10.sup.-2                                                                           10.sup.0                                                                           10.sup.-2                                                                           60V 20V                                            __________________________________________________________________________     -hs CAN-S 58 ± 3                                                                          32 ± 3                                                                           80 ± 2                                                                          33 ± 7                                                                           79 ± 5                                                                          52 ± 5                                         17-2 60 ± 3 34 ± 8 77 ± 3 37 ± 5 87 ± 3 43 ± 2                                             +hs CAN-S 69 ± 4 41 ± 4 77 ± 2                                       25 ± 9 74 ± 5 58 ± 6                    (3 hrs) 17-2 71 ± 4 37 ± 3 76 ± 5 26 ± 3 78 ± 3 67 ±                                       5                                                +hs CAN-S 66 ± 2 56 ± 8 79 ± 4 33 ± 2 84 ± 3 63 ± 3                                         (24 hr) 17-2 65 ± 3 42 ± 6 76 ±                                      3 41 ± 5 85 ± 2 60 ± 6                  -hs CAN-S 51 ± 4 39 ± 5 72 ± 5 33 ± 7 87 ± 3 52 ± 5                                          rsh; 17-2 57 ± 3 39 ± 5 74 ± 5                                      29 ± 4 82 ± 4 53 ± 6                    +hs CAN-S 72 ± 4 48 ± 3 66 ± 2 60 ± 3 80 ± 4 58 ± 6                                         (3 hr) rsh; 17-2 68 ± 4 46 ± 6 78                                       ± 2 49 ± 4 83 ± 1 50 ±             __________________________________________________________________________                                     5                                               .sup.a Olfactory acuity and shock reactivity were assayed in untrained         flies with the methods of Boynton and Tully (1992) and Dura et al. (1993)      respectively (see Experimental Procedures for more details). N = 98 PIs        per group. Planned comparisons between CanS vs. mutant flies failed to         detect any significant differences with any heatshock regimen.                 *10.sup.0 is manual concentration and corresponds to 10.sup.-3 for bubble      (see Tully et al., 1994).                                                

FIG. 12 shows that induction of hs-dCREB2-b completely abolishes one-day memory after spaced training in radish; 17-2 "double mutants." Since radish is known to disrupt ARM, a clear view of the effect of hs-dCREB2-b on LTM was obtained in radish;17-2 flies. One-day retention after spaced training was assayed in rsh;17-2 double mutants and in 17-2 and rsh single-gene mutants as controls. Flies were trained in the absence of heat-shock (hatched bars) or three hours after heat-shock (striped bars) and stored at 18° C. during the retention interval. As usual, induction of hs-dCREB2-b produced significantly lower one-day memory after spaced training in 17-2 flies (P<0.001). The heat-shock regimen, however, had no effect on such memory in radish mutants (P=0.52), which reflects only the presence of LTM (see Tully et al., 1994). In contrast, heat-shock produced significantly lower scores in rash;17-2 double mutants (P<0.001), which were not different from zero (P=0.20). N=6 PIs per group.

The following materials and methods were used in the work described in Examples 5 and 6.

Pavlovian Learning and Memory and Testing

During one training session, a group of about 100 flies was exposed sequentially to two odors [either octanol (OCT) or methylcyclohexanol (MCH)] for 60s with 45-s rest intervals after each odor presentation. During exposure to the first odor, flies received twelve 1.5-s pulses of 60 V DC with a 5-s interpulse interval.

After training, flies were transferred to food vials and stored at 18° C. for a seven-day retention interval. Conditioned odor-avoidance responses then were tested by transferring files to the choice point of a T-maze, where they were exposed simultaneously to OCT and MCH carried on converging currents of air in the distal ends of the T-maze arms and out the choice point.

Flies were allowed to distribute themselves in the T-maze arms for 120s, after which they were trapped in their respective arms, anesthetized and counted. The "percent correct" then was calculated as the number of flies avoiding the shock-paired odor (they were in the opposite T-maze arm) divided by the total number of flies in both arms. (The number of flies left at the choice point, which usually was less than 5%, were not included in this calculation.) Finally, a performance index (PI) was calculated by averaging the percent corrects of two reciprocal groups of flies--one where OCT and shock were paired, the other where MCH and shock were paired--and then by normalizing the average so that a PI=0 represented a 50:50 distribution in the T-maze and a PI=100 represented 100% avoidance of the shock-paired odor.

All behavioral experiments were designed in a balanced fashion with N=2 PIs per group collected per day; then replicated across days to generate final Ns. In all experiments, the experimenter was blind to genotype.

Statistical Analyses of Behavior Data

PIs are distributed normally (Tully and Gold 1993). Consequently, untransformed (raw) data were analyzed parametrically with JMP3.01 statistical software (SAS Institute Inc., Cary NC). Negative accelerating exponential Gompertz (growth) functions (see Lewis 1960) were fit to the data in FIGS. 13A and 13B via nonlinear least squares with iteration.

Example 5 Effect on Long Term Memory of Repeated Training Sessions

Seven-day memory retention (a measure of long term memory) in wild-type (Can-S) flies is induced incrementally by repeated training sessions. An automated version of a discriminative classical conditioning procedure was used to electroshock flies during exposure to one odor (CS+) but not to a second odor (CS-). Seven days after one or more training sessions, memory retention of conditioned odor avoidance responses was quantified in a T-maze, where flies were presented the CS+ and CS- simultaneously for 120s.

In FIG. 13A, long term memory as a function of the number of training sessions is indicated by open circles. One training session produced a mean performance index (PI±SEM; Note 1) near zero. Additional training sessions with a 15-min rest interval between each, however, yielded a steady increase in mean PIs with a maximum of 39 after ten training sessions. Ten additional training sessions produced similar performance. A nonlinear "growth" function (solid line) was fit to the individual PIs using an iterative least squares method. N=13, 6, 6, 6, 13, 7, 7, 7, 7, 6, 7 and 7 PIs for groups receiving 1-10, 15 and 20 training sessions, respectively.

Example 6 Effect on Long Term Memory of the Rest Interval Between Each Training Session

Seven-day memory retention (a measure of long term memory) in wild-type (Can-S) flies is induced incrementally by the rest interval between each training session. As described in Example 5, an automated version of a discriminative classical conditioning procedure was used to electroshock flies during exposure to one odor (CS+) but not to a second odor (CS-). Seven days after one or more training sessions, memory retention of conditioned odor avoidance responses was quantified in a T-maze, where flies were presented the CS+ and CS- simultaneously for 120s.

In FIG. 13B, long term memory as a function of the rest interval is indicated by open circles. Ten training sessions with no rest interval between each (massed training) produced a mean PI near zero. Increasing the rest interval between each of ten training sessions yielded a steady increase in mean PIs with a maximum of 34 for a 10-min rest interval. Rest intervals up to ten minutes longer produced similar performance. A nonlinear growth function (solid line) was fit to the data as above. N=12, 6, 6, 6, 6, 13, 7, 7, 7, 7, 7, 7 and 7 PIs for groups receiving 0-10, 15 and 20 min of rest between each training session.

The following materials and methods were used in the work described in Examples 7-10.

Isolating Transgenic Flies

EcoRI restriction sites were added (using PCR) just 5' to the putative translation initiation site and just 3' to the translation termination site in the dCREB2-a CDNA. This fragment was sequenced and subcloned into CaSpeR hs43, a mini-white transformation vector which contains the hsp70 promoter, in the orientation so that the dCREB2-a open reading frame is regulated by the hsp70 promoter. Germ-line transformation was accomplished by injecting into isogenic w(isoCJ1) embryoes using standard techniques (Spradling and Rubin, 1982; Rubin and Spradling, 1982). By injecting DNA into the relatively homogeneous genetic background of w(isoCJ1), outcrossing of the resulting germ-line transformants to equilibrate (heterogeneous) genetic backgrounds was not necessary. Two transgenic lines, C28 and C30, each with one independent P-element insertion were generated and characterized. They appeared normal in general appearance, fertility and viability. Flies homozygous for the C28 or C30 transgene were bred and used for all experiments.

Heat Shock Regimen

For heat-shock induction, flies were collected within two days of eclosion, placed in glass bottles in groups of about 600, and incubated overnight at 25° C. and 70% relative humidity. The next day, three hours before training, groups of approximately 100 flies were transferred to foam-stoppered glass shell vials containing a strip of filter paper to absorb excess moisture. The vials then were submerged in a 37° C. water bath until the bottom of the foam stopper (inside the vial) was below the surface of the water, thereby insuring that the flies could not escape heat-shock. The vial remained submerged for 30 min, after which the flies were transferred to a standard food vial for a 3-hr recovery period at 25° C. and 70% relative humidity. Training began immediately after the recovery period.

Statistical Analyses of Behavior Data

PIs from the three strains (Can-S, C28 and C30) and six training-regiments (1×+hs, 2'massed+hs, 10×massed+hs, 1×-hs, 2×massed-hs and 10×massed-hs) were subjected to a TWO-WAY ANOVA with STRAIN (F.sub.(2,102) =48.34; P<0.001) and TRAINing-regimen (F.sub.(5,102) =25.47, P<0.001) as main effects and STRAIN×TRAIN (F.sub.(10,102) =5.85, P<0.001) as the interaction term. Since preliminary experiments preceded all of the experiments summarized herein, all pairwise comparisons were planned. To maintain an experimentwise error rate of alpha=0.05, the individual comparisons summarized in FIG. 15B were judged significant if P<0.002 (Sokal and Rohlf 1981).

Example 7 A Molecular Switch for the Formation of Long Term Memory

FIG. 14 presents a conceptual method of a molecular switch for the formation of LTM, based on differential regulation of CREB isoforms with opposing functions.

Immediately after one training session, the relevant CREB activators and repressors are induced. Their combined functions (rather than molecular concentrations) are equivalent and yielded no net effect of CREB activators. Thus, repeated sessions of massed training (no rest interval) never induce LTM (see FIG. 15A). CREB repressors functionally inactivate faster than CREB activators, however, yielding an increasing net effect of CREB activators (ΔC) with time (see FIG. 13B). If ΔC is positive at the end of a particular rest interval during spaced training, then CREB activators are free to initiate downstream events involved with the formation of LTM. Usually, ΔC after one training session is not large enough to yield much LTM. Thus, repeated spaced training sessions serve to increase ΔC incrementally eventually to produce maximal LTM (see FIG. 13A).

Experimental verification of two predictions from this model involving CREB as a molecular switch for long term memory formation is shown in FIGS. 15A-15C and discussed in Examples 8-10.

Example 8 Effect on Long Term Memory of Having Equal Amounts of CREB Activators and Repressors Immediately After One Training Session

The model of a molecular switch for LTM predicts that the functional effects of all CREB activators and repressors are equal immediately after one training session (ΔC=0). If no rest interval occurred between additional training sessions (massed training), then functional CREB activator would not accumulate, thereby preventing the induction of downstream events required for LTM induction.

To test this notion, wild-type (Can-S) flies were subjected to 48, instead of the usual 10 (see FIG. 15B), massed training sessions (48× massed) or, as a positive control, to 10 spaced training sessions with a 15-min rest interval (10× spaced). Seven-day memory after such massed training was near zero (FIG. 15A), while that after spaced training was near its usual maximum value (see FIG. 13A). Thus, nearly five times the usual amount of massed training still did not induce LTM. N=6 PIs for each group.

PIs from two groups (10× spaced or 48× massed) of wild-type (Can-S) files were subjected to a ONE-WAY ANOVA with GROUP (F.sub.[10] =51.13; P<0.001) as the main effect. A subsequent planned comparison revealed that the mean PI of the 48× massed group did not differ significantly from zero (t.sub.[10] =1.66; P=0.127).

Example 9 Effect on Long Term Memory of Increasing Amounts of CREB Activator

The model of a molecular switch for LTM predicts that experimentally increasing the amount of CREB activator will eliminate the requirements for at least 10 repeated training sessions with a 10-min rest interval between each to produce maximal LTM.

To test this idea, two transgenic lines (C28 and C30) carrying an inducible hsp-dCREB2-a activator construct inserted into different cytological locations were generated. Different groups of flies from these two transgenic lines were subjected, along with wild-type (Can-S) files, to 1 (1×) 2 (2×) or (10×) massed training sessions three hours after heat-shock induction of the transgene (induced) or in the absence of heat-shock (uninduced).

Without heat-shock, seven-day memory in all three strains did not differ from zero after one, two or ten massed training sessions (all Ps>0.002;). With heat-shock, seven-day memory in wild-type flies remained near zero in each massed training group (all Ps>0.002). In contrast, seven-day memory was significant (near the maximum of 35) after ten massed sessions in both the C28 and C30 transgenic lines (all Ps<0.0001). Moreover, seven-day memory after one training session was similar to that after ten training sessions in both C28 (P=0.89) and C30 (P=0.89) transgenic flies. Thus, maximum LTM was induced after just one training session in transgenic flies expressing abnormally high levels of CREB activator. N=10, 4 and 6 PIs for each group of Can-S, C28 and C30, respectively.

Example 10 Olfactory Acuity and Shock Reactivity

Odor avoidance responses to OCT or to MCH were quantified with the method of Boynton and Tully (1992) given a choice between an odor and air. The odors are naturally aversive, and flies usually chose air and avoided the T-maze arm containing the odor. After 120s, the flies were trapped in their respective arms of the T-maze, anesthetized and counted. A PI was calculated as a normalized percent correctly avoiding the odor (cf. Example 5). PIs from these two strains and two odor-groups (OCT and MCH) were subjected to a TWO-WAY ANOVA with STRAIN (F.sub.(1,12) =1.57, P=0.23) and ODOR (F.sub.(1,12) =0.07, P=0.80) as main effects and DRUG×ODOR (F.sub.(1,12) =0.15, P=0.71) as the interaction term. The two subsequent planned comparisons were judged significant if P<0.025.

Shock reactivity was quantified with the method of Dura et al. (1993) in wild-type (Can-S) flies, or in a transgenic line (C28) carrying an inducible hsp-dCREB2-a construct, three hours after a 30-min heat shock at 37° C. Briefly, flies were placed in a T-maze and given a choice between an electrified grid (60 V DC) in one T-maze arm and an unconnected grid in the other. After 120s, the flies were trapped in their respective T-maze arms, anesthetized and counted. A PI was calculated as for olfactory acuity. PIs from these two strains were subjected to a ONE-WAY ANOVA with STRAIN (F.sub.(1, 6) =13.03, P=0.01) as the main effect.

Naive avoidance responses to odors or to shock three hours after heat-shock did not differ between wild-type (Can-S) versus transgenic (C28) flies for the two odorants (MCH and OCT) used for conditioning experiments (P=0.27, 0.55, respectively). N=4 PIs per group. Naive shock avoidance responses three hours after training for transgenic flies were slightly lower than those for wild-flies (P=0.01). N=4 PIs per group.

Examples 11-13 pertain to the Drosophila nitric oxide synthase work.

Example 11 Low Stringency Hybridization to a Phage Library of the Drosophila Genome and Screening of Drosophila cDNA Library

6×10⁴ plaques of a genomic Drosophila λDASH library with the 1.3 kb Bgl II fragment of rat neuronal NOS CDNA (residues 3282-4573) under low stringency conditions of 40% formamide were screened as described in W. M. McGinnis et al., Nature 308:428 (1984). Fifty positive phage were purified and grouped based on inter se hybridization. The contig containing the 2.4R fragment of dNOS was comprised of 15 phage clones. Regions of cross-hybridization to the rat probe were identified, subcloned and three of them were sequenced. The other two did not contain sequences homologous to any protein in the database. A Drosophila head cDNA library (a gift from P. Salvaterra) was screened with the 2.4R fragment isolated from phage clone λ8.11 in standard conditions. All phage purification and cloning steps were done with standard methods (J. Sambrook, E. F. Fritsch, T. Maniatis, Molecular cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989). cDNA fragments were subcloned into Bluescript (Stratagene) and sequenced on both strands with Sequenase 2.0 (USB).

Example 12 Activity of Drosophila Nitric Oxide Synthase (dNOS)

The expression construct for activity assays contained dNOS cDNA (with an XbaI site engineered immediately upstream of the ATG codon) cloned into the XbaI and SmaI sites of the pCGN expression vector [M. Tanaka and W. Herr, Cell, 60:375 (1990)]. 293 human kidney cells were transfected with 15 μg of the dNOS construct, or vector DNA, and precipitated with calcium phosphate as described in [M. J. Imperiale, L. T. Feldman and J. R. Nevins, Cell, 35:127 (1983)]. Cells were collected 2 days later and protein extracts were prepared as described in [J. Sambrook, E. F. Fritsch, T. Maniatis, Molecular cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989)].

The fusion protein for raising anti-DNOS antibodies was made by cloning a 0.29 kb Eam1105I-SacI fragment of dNOS cDNA (this fragment codes for 97 N-terminal amino acids of dNOS ORF) into EcoRI site of PGEX-KG [K. Guan and J. E. Dixon, Anal. Biochem., 192:262 (1991)]. The fusion protein was expressed in BL21 E. coli strain and purified over Glutathione-Sepharose columns (Pharmacia) as described in [G. J. Hannon, D. Demetrick, D. Beach, Genes & Dev., 7:2378 (1993)]. Immunization of rabbits, and serum preparation, was done by Hazleton Research Products, Inc. (Denver). The DNOS protein was detected on Western blots using a 1:500 dilution of rabbit serum, and cross-reacting bands were visualized with anti-rabbit alkaline phosphatase conjugate (Promega) according to the protocol provided.

The enzymatic assay was done essentially as described previously (D. Bredt and S. Snyder, Proc. Natl. Acad. Sci. USA, 87:682 (1990)]. A 100 ml reaction mixture containing 25 μl (50-100 μg) of soluble protein extract, 50 mM Hepes pH 7.4, 3 μM FAD, 3 μM FMN, 10 μM tetrahydrobiopterin (ICN), 1 mM DTT, 0.8 mM CaCl₂, 1 mM NADPH, 10 μg/ml calmodulin, 2μl of [³ H]L-arginine (35.7 Ci/mmol, NEN) and 50 mM L-valine in was incubated for 60 minutes at 37° C. The reaction was stopped with 0.5 ml 20 mM Hepes pH 5.5, 2 mM EDTA, 2 mM EGTA, loaded on 0.5 ml Dowex AG 50WX-8 (Na⁺ form) column and eluted with 3×0.5 ml of the stop buffer. Radioactivity present in the eluent was quantified in a scintillation counter.

FIGS. 17A-17B show the expression of DNOS enzymatic activity in 293 kidney cells. FIG. 17A shows the results of a Western blot analysis of protein extracts from 293 cells transfected with vector alone (lane 293+vector) or with dNOS CDNA construct (lane 293+dNOS). 25 μg of soluble protein extracts was resolved on 7.5% polyacrylamide gel, transferred to nitrocellulose membrane and treated with anti-DNOS antibody. The arrow indicates the position of the DNOS protein. Positions of molecular weight markers (in kD) are shown on the left.

FIG. 17B shows siginificant DNOS enzyme activity measured in 293 cell extracts by conversion of ³ H-L-arginine to ³ H-L-citrulline. Enzymatic activity was detected only in cells transfected with dNOS cDNA construct (groups B-D) and is presented as specific activity (pmol of citrulline/mg/min.). The DNOS activity also was measured in the presence of 1 mM EGTA without exogenous Ca²⁺ or calmodulin (group C), or in the presence of 100 mM L-NAME (group D). N=4 reactions per group.

Example 13 Splicing Pattern of dNOS

Heads and bodies of adult flies were separated on sieves. Total RNA was isolated by the guanidinium isothiocyanate method [P. Chomczynski and N. Sacchi, Anal. Biochem., 162:156 (1987)]. Poly(A)⁺ RNA selection, Northern blot and hybridization were done with standard methods (J. Sambrook, E. F. Fritsch, T. Maniatis, Molecular cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989)]. The blot was hybridized with random-primed dNOS cDNA (10⁶ cpm/ml), washed in 0.1×SSC and 0.1% SDS at 65° C. and exposed to X-ray film for 72 hours. Two 25-mer primers [corresponding to residues 1374-1399 (the top primer) and 1793-1817 (the bottom primer) in the dNOS sequence] were used to amplify fragments of two dNOS splice products. Each RT-PCR reaction contained 30 ng of poly(A)⁺ head RNA. In the first stage (RT), 90 ng of the bottom primer and 5 U of rTth polymerase (Perkin-Elmers) were added and the mixture was incubated in the MJ Research Minicycler™ in the following sequence of conditions: 95°/1 minute, 67°/45 seconds, 70°/13 minutes. The second stage (PCR) was carried out as follows: 94°/45 seconds, 63°/45 seconds, 70°/90 seconds and was repeated for 35 cycles. Products of the reaction were analyzed on a denaturing polyacrylamide (8%) gel. Bands of interest were isolated, reamplified, cloned into pCR1000 (InVitrogen) and sequenced with Sequenase kit (USB).

Northern blot analysis of dNOS expression in adult flies shows a 5.0 kb dNOS transcript present in heads (FIG. 18A). Each lane contained 10 mg of poly (A)⁺ mRNA isolated from Drosophila heads (H) or bodies (B). The Northern blot was hybridized with the dNOS cDNA as described above (30). Positions of size markers (in kb) are shown on the left. The blot was overprobed with myosin light chain (MLC) (41) as a standard for RNA concentration.

FIG. 18B shows that the dNOS gene expresses two alternatively spliced mRNA species. RT-PCR reactions were performed on poly(A)⁺ mRNA isolated from Drosophila heads and were resolved on 8% polyacrylamide gel. Arrows indicate the positions of DNA fragments of expected sizes: the 444 bp long-form fragment and the 129 bp short-form fragment (lane +RNA). Other bands present in this lane are artifacts from heteroduplexes that failed to denature. Poly(A)⁺ mRNA was omitted from the control reaction (lane -RNA), which otherwise was done in identical conditions. Size markers (kb ladder) are shown in the middle lane (KB).

Equivalents

Those skilled in the art will know, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. These and all other equivalents are intended to be encompassed by the following claims.

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15. G. A. Bohme, C. Bon, J.-M. Stutzman, A. Doble, J.-C. Blanchard, Eur. J. Pharmacol. 199, 379 (1991); E. M. Schuman, D. V. Madison, Science 254, 1503 (1991); T. J. O'Dell, R. D. Hawkins, E. R. Kandel, O. Arancio, Proc. Natl. Acad. Sci. USA 88, 11285 (1991); K. Shibuki, D. Okada, Nature 349, 326 (1991); J. E. Haley, G. L. Wilcox, P. F. Chapman, Neuron 8, 211 (1992); M. Zhuo, S. A. Small, E. R. Kandel, R. D. Hawkins, Science 260, 1946 (1993); M. Zhuo, E. R. Kandel, R. D. Hawkins, NeuroReport 5, 1033 (1994).

16. P. F. Chapman, C. M. Atkins, M. T. Allen, J. E. Haley, J. E. Steinmetz, NeuroReport3, 567 (1992); C. Holscher, S. P. R. Rose, Neurosci. Lett. 145, 165 (1992); G. A. Bohme, et al., Proc. Natl. Acad. Sci. USA 90, 9191 (1993); N. S. Rickard, K. T. Ng, M. E. Gibbs, Behav. Neurosci. 108, 640-644 (1994).

17. P. L. Huang, T. M. Dawson, D. S. Bredt, S. H. Snyder, M. C. Fishman, Cell 75, 1273-1286 (1993).

18. T. J. O'Dell, et al., Science 265, 542-546 (1994).

19. We have screened 6×10⁴ plaques of a genomic Drosophila λDASH library with the 1.3 kb Bgl 11 fragment of rat neuronal NOS cDNA (residues 3282-4573) under low stringency conditions of 40% formamide as described in W. M. McGinnis et al., Nature 308, 428 (1984). Fifty positive phage were purified and grouped based on inter se hybridization. The contig containing the 2.4R fragment of dNOS was comprised of 15 phage clones. Regions of cross-hybridization to the rat probe were identified, subcloned and three of them were sequenced. The other two did not contain sequences homologous to any protein in the database. A Drosophila head cDNA library (a gift from P. Salvaterra) was screened with the 2.4R fragment isolated from phage clone λ8.11 in standard conditions. All phage purification and cloning steps were done with standard methods (J. Sambrook, E. F. Fritsch, T. Maniatis, Molecular cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989). cDNA fragments were subcloned into Bluescript (Stratagene) and sequenced on both strands with Sequenase 2.0 (USB). Single-letter abbreviations for the amino acid residues are as follows: A, Ala;C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gin; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr.

20. D. R. Cavener, Nucleic Acids Res 15, 1353-1361 (1987).

21. K. T. O'Neil, W. F. DeGrado, Trends Biochem. Sci. 15, 59-64 (1990).

22. R. B. Pearson, B. E. Kemp, Meth. Enzymol. 200, 62-81 (1991).

23. R. G. Franks, S. T. Crews, Mech. Dev. 45, 269 (1994); H.-P. Gerber, et al., Science 263, 808 (1994); M. Reguiski, N. McGinnis, R. Chadwick, W. McGinnis, EMBO J. 6, 767(1987).

24. The expression construct for activity assays contained dNOS cDNA (with an XbaI site engineered immediately upstream of the ATG codon) cloned into the XbaI and SmaI sites of the pCGN expression vector [M. Tanaka and W. Herr, Cell 60, 375 (1990)]. 293 human kidney cells were transfected with 15 μg of the dNOS construct, or vector DNA, and precipitated with calcium phosphate as described in [M. J. Imperiale, L. T. Feldman and J. R. Nevins, Cell 35, 127 (1983)]. Cells were collected 2 days later and protein extracts were prepared as described in [J. Sambrook, E. F. Fritsch, T. Maniatis, Molecular cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989)].

25. The fusion protein for raising anti-DNOS antibodies was made by cloning a 0.29 kb Eam1105I-SacI fragment of dNOS cDNA (this fragment codes for 97 N-terminal amino acids of dNOS ORF) into EcoRI site of pGEX-KG [K. Guan and J. E. Dixon Anal. Biochem. 192, 262 (1991)]. The fusion protein was expressed in BL21 E. coli strain and purified over Glutathione-Sepharose columns (Pharmacia) as described in [G. J. Hannon, D. Demetrick, D. Beach, Genes & Dev., 7, 2378 (1993)]. Immunization of rabbits, and serum preparation, was done by Hazleton Research Products, Inc. (Denver). The DNOS protein was detected on Western blots using a 1:500 dilution of rabbit serum, and cross-reacting bands were visualized with anti-rabbit alkaline phosphatase conjugate (Promega) according to the protocol provided.

26. The enzymatic assay was done essentially as described previously (D. Bredt and S. Snyder, Proc. Natl. Acad. Sci. USA, 87, 682 (1990)]. A 100 ml reaction mixture containing 25 μl (50-100 μg) of soluble protein extract, 50 mM Hepes pH 7.4, 3 μM FAD, 3 μM FMN, 10 μM tetrahydrobiopterin (ICN), 1 mM DTT, 0.8 mM CaCl₂, 1 mM NADPH, 10 μg/ml calmodulin, 2 μl of [³ H]L-arginine (35.7 Ci/mmol, NEN) and 50 mM L-valine in was incubated for 60 min. at 37° C. The reaction was stopped with 0.5 ml 20 mM Hepes pH 5.5, 2 mM EDTA, 2 mM EGTA, loaded on 0.5 ml Dowex AG 50WX-8 (Na⁺ form) column and eluted with 3×0.5 ml of the stop buffer. Radioactivity present in the eluent was quantified in a scintillation counter.

27. R. Iyengar, D. J. Steuhr, M. A. Marletta, Proc. Natl Acad. Sci. USA 84, 6369-6373 (1987).

28. D. S. Bredt, S. H. Snyder, Proc. Natl. Acad. Sci. USA 87, 682-685 (1990).

29. D. D. Rees, R. M. J. Palmer, R. Schulz, H. F. Hodson, S. Moncada, Br. J. Pharmacol. 101, 746-752 (1990).

30. Heads and bodies of adult flies were separated on sieves. Total RNA was isolated by the guanidinium isothiocyanate method [P. Chomczynski and N. Sacchi, Anal. Biochem. 162, 156 (1987)]. Poly(A)⁺ RNA selection, Northern blot and hybridization were done with standard methods (J. Sambrook, E. F. Fritsch, T. Maniatis, Molecular cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989). The blot was hybridized with random-primed dNOS cDNA (10⁶ cpm/ml), washed in 0.1×SSC and 0.1% SDS at 65° C. and exposed to X-ray film for 72 hrs. We have used two 25-mer primers [corresponding to residues 1374-1399 (the top primer) and 1793-1817 (the bottom primer) in the dNOS sequence] to amplify fragments of two dNOS splice products. Each RT-PCR reaction contained 30 ng of poly(A)⁺ head RNA [J. C. P. Yin et al., Mol. Cell Biol., submitted]. In the first stage (RT), 90 ng of the bottom primer and 5 U of rTth polymerase (Perkin-Elmers) were added and the mixture was incubated in the MJ Research Minicycler™ in the following sequence of conditions: 95°/1 min., 67°/45sec, 70°/13 min. The second stage (PCR) was carried out as follows: 94°/45 sec., 63°/45 sec., 70°/90 sec. and was repeated for 35 cycles. Products of the reaction were analyzed on a denaturing polyacrylamide (8%) gel. Bands of interest were isolated, re-amplified, cloned into pCR1000 (InVitrogen) and sequenced with Sequenase kit (USB).

31. T. Ogura, T. Yokoyama, H. Fujisawa, Y. Kurashima, H. Esumi, Biochem. Biophys. Res. Commun. 193, 1014-1022 (1993).

32. M. W. Radomski, J. F. Martin, S. Moncada, Philos. Trans. R. Soc. Lond. B Biol. Sci. 334, 129-133 (1992).

33. M. R. Elphick, I. C. Green, M. O'Shea, Brain Res. 619, 344-346 (1993).

34. J. M. C. Ribeiro, R. H. Nussenzweig, FEBS Let. 330, 165-168 (1993).

35. R. Elofsson, M. Carlberg, L. Moroz, L. Nezlin, D. Sakharov, NeuroReport 4, 279-282 (1993).

36. A. Gelperin, Nature 369, 61-63 (1994).

37. T. M. Dawson, D. S. Bredt, M. Fotuhi, P. M. Hwang, S. H. Snyder, Proc. Natl. Acad. Sci. USA 88, 7797 (1991); B. T. Hope, G. J. Michael, K. M. Knigge, S. R. Vincent, Proc. Natl. Acad. Sci. USA 88, 2811 (1991).

38. U. Muller, E. Buchner, Naturwissenschaft 80, 524-526 (1993).

39. J. C. P. Yin, et al., Cell, in press (1994); P. Drain, E. Folkers, W. G. Quinn, Neuron 6, 71(1991).

40. P. A. Karplus, M. J. Daniels, J. R. Herriott, Science 251, 60-66 (1991).

41. V. P. Parker, S. Falkenthal, N. Davidson, Mol. Cell Biol. 5, 3058 (1985).

    __________________________________________________________________________     #             SEQUENCE LISTING                                                    - -  - - (1) GENERAL INFORMATION:                                              - -    (iii) NUMBER OF SEQUENCES: 24                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1083 base - #pairs                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                          (A) DESCRIPTION: /desc - #= "cDNA and PCR analysis"                   - -     (ix) FEATURE:                                                                   (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..1080                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                - - ATG GAC AAC AGC ATC GTC GAG GAG AAC GGC AA - #C TCG TCG GCG GCA         TCG       48                                                                     Met Asp Asn Ser Ile Val Glu Glu Asn Gly As - #n Ser Ser Ala Ala Ser             1               5 - #                 10 - #                 15               - - GGC TCC AAT GAC GTG GTC GAT GTC GTT GCC CA - #A CAG GCG GCG GCA GCG            96                                                                        Gly Ser Asn Asp Val Val Asp Val Val Ala Gl - #n Gln Ala Ala Ala Ala                         20     - #             25     - #             30                   - - GTG GGC GGC GGC GGT GGA GGA GGA GGA GGC GG - #C GGC GGC GGT GGT AAC           144                                                                        Val Gly Gly Gly Gly Gly Gly Gly Gly Gly Gl - #y Gly Gly Gly Gly Asn                     35         - #         40         - #         45                       - - CCC CAG CAG CAG CAA CAG AAC CCA CAA AGT AC - #A ACG GCC GGC GGT CCA           192                                                                        Pro Gln Gln Gln Gln Gln Asn Pro Gln Ser Th - #r Thr Ala Gly Gly Pro                 50             - #     55             - #     60                           - - ACG GGT GCG ACG AAC AAC GCC CAG GGA GGC GG - #A GTG TCC TCC GTG CTG           240                                                                        Thr Gly Ala Thr Asn Asn Ala Gln Gly Gly Gl - #y Val Ser Ser Val Leu             65                 - # 70                 - # 75                 - # 80        - - ACC ACC ACC GCC AAC TGC AAC ATA CAA TAC CC - #C ATC CAG ACG CTG GCG           288                                                                        Thr Thr Thr Ala Asn Cys Asn Ile Gln Tyr Pr - #o Ile Gln Thr Leu Ala                             85 - #                 90 - #                 95               - - CAG CAC GGA CTG CAG GTG AGC ATT TGG GGA CC - #G GGT GCT TGG TGT CAA           336                                                                        Gln His Gly Leu Gln Val Ser Ile Trp Gly Pr - #o Gly Ala Trp Cys Gln                        100      - #           105      - #           110                   - - CTG TCG AGT GTC AGG TGT TAC GGA TCC CAG CC - #A GAA GTG GCT ACC AAG           384                                                                        Leu Ser Ser Val Arg Cys Tyr Gly Ser Gln Pr - #o Glu Val Ala Thr Lys                    115          - #       120          - #       125                       - - GAT GTG CAG TCC GTG ATA CAG GCC AAT CCC TC - #G GGA GTC ATA CAG ACA           432                                                                        Asp Val Gln Ser Val Ile Gln Ala Asn Pro Se - #r Gly Val Ile Gln Thr                130              - #   135              - #   140                           - - GCA GCT GGA ACC CAG CAG CAG CAA CAG GCG CT - #G GCC GCC GCC ACA GCG           480                                                                        Ala Ala Gly Thr Gln Gln Gln Gln Gln Ala Le - #u Ala Ala Ala Thr Ala            145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - ATG CAG AAG GTG GTC TAC GTG GCC AAG CCG CC - #G AAC TCG ACG GTC         ATC      528                                                                     Met Gln Lys Val Val Tyr Val Ala Lys Pro Pr - #o Asn Ser Thr Val Ile                           165  - #               170  - #               175               - - CAC ACG ACG CCT GGC AAT GCA GTG CAA GTG CG - #T AAC AAA ATC CCT CCA           576                                                                        His Thr Thr Pro Gly Asn Ala Val Gln Val Ar - #g Asn Lys Ile Pro Pro                        180      - #           185      - #           190                   - - ACC TTT CCA TGT AAG ATC AAG CCC GAA CCG AA - #C ACG CAG CAC CCG GAG           624                                                                        Thr Phe Pro Cys Lys Ile Lys Pro Glu Pro As - #n Thr Gln His Pro Glu                    195          - #       200          - #       205                       - - GAC AGC GAC GAG AGT CTG TCG GAC GAC GAT TC - #C CAG CAC CAC CGC AGC           672                                                                        Asp Ser Asp Glu Ser Leu Ser Asp Asp Asp Se - #r Gln His His Arg Ser                210              - #   215              - #   220                           - - GAG CTG ACG CGA CGG CCG TCG TAC AAT AAG AT - #C TTC ACC GAG ATC AGC           720                                                                        Glu Leu Thr Arg Arg Pro Ser Tyr Asn Lys Il - #e Phe Thr Glu Ile Ser            225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - GGT CCG GAC ATG AGC GGC GCA TCG CTT CCC AT - #G TCC GAC GGC GTG         CTC      768                                                                     Gly Pro Asp Met Ser Gly Ala Ser Leu Pro Me - #t Ser Asp Gly Val Leu                           245  - #               250  - #               255               - - AAT TCC CAG CTG GTG GGG ACC GGA GCG GGG GG - #C AAT GCG GCG AAC AGC           816                                                                        Asn Ser Gln Leu Val Gly Thr Gly Ala Gly Gl - #y Asn Ala Ala Asn Ser                        260      - #           265      - #           270                   - - TCC CTG ATG CAA TTG GAT CCC ACG TAC TAC CT - #G TCC AAT CGG ATG TCC           864                                                                        Ser Leu Met Gln Leu Asp Pro Thr Tyr Tyr Le - #u Ser Asn Arg Met Ser                    275          - #       280          - #       285                       - - TAC AAC ACC AAC AAC AGC GGG ATA GCG GAG GA - #T CAG ACC CGT AAG CGC           912                                                                        Tyr Asn Thr Asn Asn Ser Gly Ile Ala Glu As - #p Gln Thr Arg Lys Arg                290              - #   295              - #   300                           - - GAG ATC CGG CTG CAG AAG AAC AGG GAG GCG GC - #G CGT GAG TGC CGG CGC           960                                                                        Glu Ile Arg Leu Gln Lys Asn Arg Glu Ala Al - #a Arg Glu Cys Arg Arg            305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - AAG AAG AAG GAG TAC ATC AAG TGC CTG GAG AA - #T CGA GTG GCG GTG         CTA     1008                                                                     Lys Lys Lys Glu Tyr Ile Lys Cys Leu Glu As - #n Arg Val Ala Val Leu                           325  - #               330  - #               335               - - GAG AAC CAA AAC AAA GCG CTC ATC GAG GAG CT - #G AAG TCG CTC AAG GAG          1056                                                                        Glu Asn Gln Asn Lys Ala Leu Ile Glu Glu Le - #u Lys Ser Leu Lys Glu                        340      - #           345      - #           350                   - - CTC TAT TGT CAG ACC AAG AAC GAT TGA    - #                  - #                1083                                                                      Leu Tyr Cys Gln Thr Lys Asn Asp                                                        355          - #       360                                              - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 360 amino - #acids                                                 (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                - - Met Asp Asn Ser Ile Val Glu Glu Asn Gly As - #n Ser Ser Ala Ala Ser         1               5 - #                 10 - #                 15               - - Gly Ser Asn Asp Val Val Asp Val Val Ala Gl - #n Gln Ala Ala Ala Ala                    20     - #             25     - #             30                   - - Val Gly Gly Gly Gly Gly Gly Gly Gly Gly Gl - #y Gly Gly Gly Gly Asn                35         - #         40         - #         45                       - - Pro Gln Gln Gln Gln Gln Asn Pro Gln Ser Th - #r Thr Ala Gly Gly Pro            50             - #     55             - #     60                           - - Thr Gly Ala Thr Asn Asn Ala Gln Gly Gly Gl - #y Val Ser Ser Val Leu        65                 - # 70                 - # 75                 - # 80        - - Thr Thr Thr Ala Asn Cys Asn Ile Gln Tyr Pr - #o Ile Gln Thr Leu Ala                        85 - #                 90 - #                 95               - - Gln His Gly Leu Gln Val Ser Ile Trp Gly Pr - #o Gly Ala Trp Cys Gln                   100      - #           105      - #           110                   - - Leu Ser Ser Val Arg Cys Tyr Gly Ser Gln Pr - #o Glu Val Ala Thr Lys               115          - #       120          - #       125                       - - Asp Val Gln Ser Val Ile Gln Ala Asn Pro Se - #r Gly Val Ile Gln Thr           130              - #   135              - #   140                           - - Ala Ala Gly Thr Gln Gln Gln Gln Gln Ala Le - #u Ala Ala Ala Thr Ala       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Met Gln Lys Val Val Tyr Val Ala Lys Pro Pr - #o Asn Ser Thr Val         Ile                                                                                              165  - #               170  - #               175              - - His Thr Thr Pro Gly Asn Ala Val Gln Val Ar - #g Asn Lys Ile Pro Pro                   180      - #           185      - #           190                   - - Thr Phe Pro Cys Lys Ile Lys Pro Glu Pro As - #n Thr Gln His Pro Glu               195          - #       200          - #       205                       - - Asp Ser Asp Glu Ser Leu Ser Asp Asp Asp Se - #r Gln His His Arg Ser           210              - #   215              - #   220                           - - Glu Leu Thr Arg Arg Pro Ser Tyr Asn Lys Il - #e Phe Thr Glu Ile Ser       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Gly Pro Asp Met Ser Gly Ala Ser Leu Pro Me - #t Ser Asp Gly Val         Leu                                                                                              245  - #               250  - #               255              - - Asn Ser Gln Leu Val Gly Thr Gly Ala Gly Gl - #y Asn Ala Ala Asn Ser                   260      - #           265      - #           270                   - - Ser Leu Met Gln Leu Asp Pro Thr Tyr Tyr Le - #u Ser Asn Arg Met Ser               275          - #       280          - #       285                       - - Tyr Asn Thr Asn Asn Ser Gly Ile Ala Glu As - #p Gln Thr Arg Lys Arg           290              - #   295              - #   300                           - - Glu Ile Arg Leu Gln Lys Asn Arg Glu Ala Al - #a Arg Glu Cys Arg Arg       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Lys Lys Lys Glu Tyr Ile Lys Cys Leu Glu As - #n Arg Val Ala Val         Leu                                                                                              325  - #               330  - #               335              - - Glu Asn Gln Asn Lys Ala Leu Ile Glu Glu Le - #u Lys Ser Leu Lys Glu                   340      - #           345      - #           350                   - - Leu Tyr Cys Gln Thr Lys Asn Asp                                                   355          - #       360                                              - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 54 amino - #acids                                                  (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                - - Arg Lys Arg Glu Ile Arg Leu Gln Lys Asn Ar - #g Glu Ala Ala Arg Glu       1               5   - #                10  - #                15                - - Cys Arg Arg Lys Lys Lys Glu Tyr Ile Lys Cy - #s Leu Glu Asn Arg Val                   20      - #            25      - #            30                    - - Ala Val Leu Glu Asn Gln Asn Lys Ala Leu Il - #e Glu Glu Leu Lys Ser               35          - #        40          - #        45                        - - Leu Lys Glu Leu Tyr Cys                                                       50                                                                          - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 54 amino - #acids                                                  (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                - - Arg Lys Arg Glu Val Arg Leu Met Lys Asn Ar - #g Glu Ala Ala Arg Glu       1               5   - #                10  - #                15                - - Cys Arg Arg Lys Lys Lys Glu Tyr Val Lys Cy - #s Leu Glu Asn Arg Val                   20      - #            25      - #            30                    - - Ala Val Leu Glu Asn Gln Asn Lys Thr Leu Il - #e Glu Glu Leu Lys Ala               35          - #        40          - #        45                        - - Leu Lys Asp Leu Tyr Cys                                                       50                                                                          - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 54 amino - #acids                                                  (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                - - Arg Lys Arg Glu Leu Arg Leu Met Lys Asn Ar - #g Glu Ala Ala Arg Glu       1               5   - #                10  - #                15                - - Cys Arg Arg Lys Lys Lys Glu Tyr Val Lys Cy - #s Leu Glu Asn Arg Val                   20      - #            25      - #            30                    - - Ala Val Leu Glu Asn Gln Asn Lys Thr Leu Il - #e Glu Glu Leu Lys Ala               35          - #        40          - #        45                        - - Leu Lys Asp Leu Tyr Cys                                                       50                                                                          - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 54 amino - #acids                                                  (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                - - Leu Lys Arg Glu Ile Arg Leu Met Lys Asn Ar - #g Glu Ala Ala Arg Glu       1               5   - #                10  - #                15                - - Cys Arg Arg Lys Lys Lys Glu Tyr Val Lys Cy - #s Leu Glu Asn Arg Val                   20      - #            25      - #            30                    - - Ala Val Leu Glu Asn Gln Asn Lys Thr Leu Il - #e Glu Glu Leu Lys Thr               35          - #        40          - #        45                        - - Leu Lys Asp Leu Tyr Ser                                                       50                                                                          - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 798 base - #pairs                                                  (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -     (ix) FEATURE:                                                                   (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..798                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                - - ATG TTA CTC GGA GAA AAT ATG TTT TCT ACT TT - #C ACA TCG TTA GAT GCT            48                                                                        Met Leu Leu Gly Glu Asn Met Phe Ser Thr Ph - #e Thr Ser Leu Asp Ala              1               5 - #                 10 - #                 15               - - GCT ACC GCT ACA ACC AAC ACC GGT GAA TTC TT - #A ATG AAT GAA TCT CCA            96                                                                        Ala Thr Ala Thr Thr Asn Thr Gly Glu Phe Le - #u Met Asn Glu Ser Pro                         20     - #             25     - #             30                   - - AGG CAA GAA GCC GGT GAC TTA ATG TTG GAT AG - #T CTG GAT TTC AAC ATT           144                                                                        Arg Gln Glu Ala Gly Asp Leu Met Leu Asp Se - #r Leu Asp Phe Asn Ile                     35         - #         40         - #         45                       - - ATG GGC GAA AAC CTG GCA GAT GAT TTC CAG AC - #C TCG GCT TCA CCA GCT           192                                                                        Met Gly Glu Asn Leu Ala Asp Asp Phe Gln Th - #r Ser Ala Ser Pro Ala                 50             - #     55             - #     60                           - - TCG GAG GAC AAG ATG ACT CCT TTC GTT GTT GA - #T ACC AAT GTT TTT GAA           240                                                                        Ser Glu Asp Lys Met Thr Pro Phe Val Val As - #p Thr Asn Val Phe Glu             65                 - # 70                 - # 75                 - # 80        - - TCC GTC TTC AAG AAC ACC GAA GAT ACC CTT CT - #A GGA GAT ATC GAC AAT           288                                                                        Ser Val Phe Lys Asn Thr Glu Asp Thr Leu Le - #u Gly Asp Ile Asp Asn                             85 - #                 90 - #                 95               - - GTT GGT ATT GTT GAC ACG GAG TTG AAG GAG AT - #G TTC GAT TTG GTT GAC           336                                                                        Val Gly Ile Val Asp Thr Glu Leu Lys Glu Me - #t Phe Asp Leu Val Asp                        100      - #           105      - #           110                   - - TCG GAA ATC AAT AAC GGC ACT CCT ATC AAG CA - #G GAA GAA AAG GAT GAT           384                                                                        Ser Glu Ile Asn Asn Gly Thr Pro Ile Lys Gl - #n Glu Glu Lys Asp Asp                    115          - #       120          - #       125                       - - TTG GAA TTT ACT TCA AGA TCC CAG TCC ACC TC - #A GCT CTC TTG TCG TCG           432                                                                        Leu Glu Phe Thr Ser Arg Ser Gln Ser Thr Se - #r Ala Leu Leu Ser Ser                130              - #   135              - #   140                           - - AAA TCG ACT TCT GCT TCT CCA GCT GAT GCT GC - #C GCT GCA TGT GCA AGT           480                                                                        Lys Ser Thr Ser Ala Ser Pro Ala Asp Ala Al - #a Ala Ala Cys Ala Ser            145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - CCT TCG TCA TCG TCT TGT AAA AGA TCC TAT TC - #T TCT GCT CAG CTA         GAA      528                                                                     Pro Ser Ser Ser Ser Cys Lys Arg Ser Tyr Se - #r Ser Ala Gln Leu Glu                           165  - #               170  - #               175               - - ACT ACG GGT TCG GAT GCT CCA AAG AAA GAT AA - #G CTG GGC TGC ACC CCT           576                                                                        Thr Thr Gly Ser Asp Ala Pro Lys Lys Asp Ly - #s Leu Gly Cys Thr Pro                        180      - #           185      - #           190                   - - TAC ACT AGA AAA CAG AGA AAC AAT CCA TTA CC - #T CCG GTC ATT CCA AAG           624                                                                        Tyr Thr Arg Lys Gln Arg Asn Asn Pro Leu Pr - #o Pro Val Ile Pro Lys                    195          - #       200          - #       205                       - - GGT CAG GAT GTT GCT TCT ATG AAA AGG GCA AG - #A AAC ACT GAG GCC GCA           672                                                                        Gly Gln Asp Val Ala Ser Met Lys Arg Ala Ar - #g Asn Thr Glu Ala Ala                210              - #   215              - #   220                           - - AGA AGA TCA AGA GCC AGA AAA ATG GAA AGA AT - #G TCC CAA CTT GAA GAA           720                                                                        Arg Arg Ser Arg Ala Arg Lys Met Glu Arg Me - #t Ser Gln Leu Glu Glu            225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - AAG TGT CAA AGC TTG TTG AAG GAA AAC GAC GA - #C TTG AAA GCT CAA         GTT      768                                                                     Lys Cys Gln Ser Leu Leu Lys Glu Asn Asp As - #p Leu Lys Ala Gln Val                           245  - #               250  - #               255               - - CAA GCT TTG AAG AAA TTA CTT GGA CAA CAA  - #                  - #               798                                                                      Gln Ala Leu Lys Lys Leu Leu Gly Gln Gln                                                    260      - #           265                                          - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 266 amino - #acids                                                 (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                - - Met Leu Leu Gly Glu Asn Met Phe Ser Thr Ph - #e Thr Ser Leu Asp Ala         1               5 - #                 10 - #                 15               - - Ala Thr Ala Thr Thr Asn Thr Gly Glu Phe Le - #u Met Asn Glu Ser Pro                    20     - #             25     - #             30                   - - Arg Gln Glu Ala Gly Asp Leu Met Leu Asp Se - #r Leu Asp Phe Asn Ile                35         - #         40         - #         45                       - - Met Gly Glu Asn Leu Ala Asp Asp Phe Gln Th - #r Ser Ala Ser Pro Ala            50             - #     55             - #     60                           - - Ser Glu Asp Lys Met Thr Pro Phe Val Val As - #p Thr Asn Val Phe Glu        65                 - # 70                 - # 75                 - # 80        - - Ser Val Phe Lys Asn Thr Glu Asp Thr Leu Le - #u Gly Asp Ile Asp Asn                        85 - #                 90 - #                 95               - - Val Gly Ile Val Asp Thr Glu Leu Lys Glu Me - #t Phe Asp Leu Val Asp                   100      - #           105      - #           110                   - - Ser Glu Ile Asn Asn Gly Thr Pro Ile Lys Gl - #n Glu Glu Lys Asp Asp               115          - #       120          - #       125                       - - Leu Glu Phe Thr Ser Arg Ser Gln Ser Thr Se - #r Ala Leu Leu Ser Ser           130              - #   135              - #   140                           - - Lys Ser Thr Ser Ala Ser Pro Ala Asp Ala Al - #a Ala Ala Cys Ala Ser       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Pro Ser Ser Ser Ser Cys Lys Arg Ser Tyr Se - #r Ser Ala Gln Leu         Glu                                                                                              165  - #               170  - #               175              - - Thr Thr Gly Ser Asp Ala Pro Lys Lys Asp Ly - #s Leu Gly Cys Thr Pro                   180      - #           185      - #           190                   - - Tyr Thr Arg Lys Gln Arg Asn Asn Pro Leu Pr - #o Pro Val Ile Pro Lys               195          - #       200          - #       205                       - - Gly Gln Asp Val Ala Ser Met Lys Arg Ala Ar - #g Asn Thr Glu Ala Ala           210              - #   215              - #   220                           - - Arg Arg Ser Arg Ala Arg Lys Met Glu Arg Me - #t Ser Gln Leu Glu Glu       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Lys Cys Gln Ser Leu Leu Lys Glu Asn Asp As - #p Leu Lys Ala Gln         Val                                                                                              245  - #               250  - #               255              - - Gln Ala Leu Lys Lys Leu Leu Gly Gln Gln                                               260      - #           265                                          - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1350 amino - #acids                                                (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                - - Met Ser Gln His Phe Thr Ser Ile Phe Glu As - #n Leu Arg Phe Val Thr       1               5   - #                10  - #                15                - - Ile Lys Arg Ala Thr Asn Ala Gln Gln Gln Gl - #n Gln Gln Gln Gln Gln                   20      - #            25      - #            30                    - - Gln Gln Leu Gln Gln Gln Gln Gln Gln Leu Gl - #n Gln Gln Lys Ala Gln               35          - #        40          - #        45                        - - Thr Gln Gln Gln Asn Ser Arg Lys Ile Lys Th - #r Gln Ala Thr Pro Thr           50              - #    55              - #    60                            - - Leu Asn Gly Asn Gly Leu Leu Ser Gly Asn Pr - #o Asn Gly Gly Gly Gly       65                  - #70                  - #75                  - #80         - - Asp Ser Ser Pro Ser His Glu Val Asp His Pr - #o Gly Gly Ala Gln Gly                       85  - #                90  - #                95                - - Ala Gln Ala Ala Gly Gly Leu Pro Ser Leu Se - #r Gly Thr Pro Leu Arg                   100      - #           105      - #           110                   - - His His Lys Arg Ala Ser Ile Ser Thr Ala Se - #r Pro Pro Ile Arg Glu               115          - #       120          - #       125                       - - Arg Arg Gly Thr Asn Thr Ser Ile Val Val Gl - #u Leu Asp Gly Ser Gly           130              - #   135              - #   140                           - - Ser Gly Ser Gly Ser Gly Gly Gly Gly Val Gl - #y Val Gly Gln Gly Ala       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Gly Cys Pro Pro Ser Gly Ser Cys Thr Ala Se - #r Gly Lys Ser Ser         Arg                                                                                              165  - #               170  - #               175              - - Glu Leu Ser Pro Ser Pro Lys Asn Gln Gln Gl - #n Pro Arg Lys Met Ser                   180      - #           185      - #           190                   - - Gln Asp Tyr Arg Ser Arg Ala Gly Ser Phe Me - #t His Leu Asp Asp Glu               195          - #       200          - #       205                       - - Gly Arg Ser Leu Leu Met Arg Lys Pro Met Ar - #g Leu Lys Asn Ile Glu           210              - #   215              - #   220                           - - Gly Arg Pro Glu Val Tyr Asp Thr Leu His Cy - #s Lys Gly Arg Glu Ile       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Leu Ser Cys Ser Lys Ala Thr Cys Thr Ser Se - #r Ile Met Asn Ile         Gly                                                                                              245  - #               250  - #               255              - - Asn Ala Ala Val Glu Ala Arg Lys Ser Asp Le - #u Ile Leu Glu His Ala                   260      - #           265      - #           270                   - - Lys Asp Phe Leu Glu Gln Tyr Phe Thr Ser Il - #e Lys Arg Thr Ser Cys               275          - #       280          - #       285                       - - Thr Ala His Glu Thr Arg Trp Lys Gln Val Ar - #g Gln Ser Ile Glu Thr           290              - #   295              - #   300                           - - Thr Gly His Tyr Gln Leu Thr Glu Thr Glu Le - #u Ile Tyr Gly Ala Lys       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Leu Ala Trp Arg Asn Ser Ser Arg Cys Ile Gl - #y Arg Ile Gln Trp         Ser                                                                                              325  - #               330  - #               335              - - Lys Leu Gln Val Phe Asp Cys Arg Tyr Val Th - #r Thr Thr Ser Gly Met                   340      - #           345      - #           350                   - - Phe Glu Ala Ile Cys Asn His Ile Lys Tyr Al - #a Thr Asn Lys Gly Asn               355          - #       360          - #       365                       - - Leu Arg Ser Ala Ile Thr Ile Phe Pro Gln Ar - #g Thr Asp Ala Lys His           370              - #   375              - #   380                           - - Asp Tyr Arg Ile Trp Asn Asn Gln Leu Ile Se - #r Tyr Ala Gly Tyr Lys       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Gln Ala Asp Gly Lys Ile Ile Gly Asp Pro Me - #t Asn Val Glu Phe         Thr                                                                                              405  - #               410  - #               415              - - Glu Val Cys Thr Lys Leu Gly Trp Lys Ser Ly - #s Gly Ser Glu Trp Asp                   420      - #           425      - #           430                   - - Ile Leu Pro Leu Val Val Ser Ala Asn Gly Hi - #s Asp Pro Asp Tyr Phe               435          - #       440          - #       445                       - - Asp Tyr Pro Pro Glu Leu Ile Leu Glu Val Pr - #o Leu Thr His Pro Lys           450              - #   455              - #   460                           - - Phe Glu Trp Phe Ser Asp Leu Gly Leu Arg Tr - #p Tyr Ala Leu Pro Ala       465                 4 - #70                 4 - #75                 4 -       #80                                                                               - - Val Ser Ser Met Leu Phe Asp Val Gly Gly Il - #e Gln Phe Thr Ala         Thr                                                                                              485  - #               490  - #               495              - - Thr Phe Ser Gly Trp Tyr Met Ser Thr Glu Il - #e Gly Ser Arg Asn Leu                   500      - #           505      - #           510                   - - Cys Asp Thr Asn Arg Arg Asn Met Leu Glu Th - #r Val Ala Leu Lys Met               515          - #       520          - #       525                       - - Gln Leu Asp Thr Arg Thr Pro Thr Ser Leu Tr - #p Lys Asp Lys Ala Val           530              - #   535              - #   540                           - - Val Glu Met Asn Ile Ala Val Leu His Ser Ty - #r Gln Ser Arg Asn Val       545                 5 - #50                 5 - #55                 5 -       #60                                                                               - - Thr Ile Val Asp His His Thr Ala Ser Glu Se - #r Phe Met Lys His         Phe                                                                                              565  - #               570  - #               575              - - Glu Asn Glu Ser Lys Leu Arg Asn Gly Cys Pr - #o Ala Asp Trp Ile Trp                   580      - #           585      - #           590                   - - Ile Val Pro Pro Leu Ser Gly Ser Ile Thr Pr - #o Val Phe His Gln Glu               595          - #       600          - #       605                       - - Met Ala Leu Tyr Tyr Leu Lys Pro Ser Phe Gl - #u Tyr Gln Asp Pro Ala           610              - #   615              - #   620                           - - Trp Arg Thr His Val Trp Lys Lys Gly Arg Gl - #y Glu Ser Lys Gly Lys       625                 6 - #30                 6 - #35                 6 -       #40                                                                               - - Lys Pro Arg Arg Lys Phe Asn Phe Lys Gln Il - #e Ala Arg Ala Val         Lys                                                                                              645  - #               650  - #               655              - - Phe Thr Ser Lys Leu Phe Gly Arg Ala Leu Se - #r Lys Arg Ile Lys Ala                   660      - #           665      - #           670                   - - Thr Val Leu Tyr Ala Thr Glu Thr Gly Lys Se - #r Glu Gln Tyr Ala Lys               675          - #       680          - #       685                       - - Gln Leu Cys Glu Leu Leu Gly His Ala Phe As - #n Ala Gln Ile Tyr Cys           690              - #   695              - #   700                           - - Met Ser Asp Tyr Asp Ile Ser Ser Ile Glu Hi - #s Glu Ala Leu Leu Ile       705                 7 - #10                 7 - #15                 7 -       #20                                                                               - - Val Val Ala Ser Thr Phe Gly Asn Gly Asp Pr - #o Pro Glu Asn Gly         Glu                                                                                              725  - #               730  - #               735              - - Leu Phe Ser Gln Glu Leu Tyr Ala Met Arg Va - #l Gln Glu Ser Ser Glu                   740      - #           745      - #           750                   - - His Gly Leu Gln Asp Ser Ser Ile Gly Ser Se - #r Lys Ser Phe Met Lys               755          - #       760          - #       765                       - - Ala Ser Ser Arg Gln Glu Phe Met Lys Leu Pr - #o Leu Gln Gln Val Lys           770              - #   775              - #   780                           - - Arg Ile Asp Arg Trp Asp Ser Leu Arg Gly Se - #r Thr Ser Asp Thr Phe       785                 7 - #90                 7 - #95                 8 -       #00                                                                               - - Thr Glu Glu Thr Phe Gly Pro Leu Ser Asn Va - #l Arg Phe Ala Val         Phe                                                                                              805  - #               810  - #               815              - - Ala Leu Gly Ser Ser Ala Tyr Pro Asn Phe Cy - #s Ala Phe Gly Gln Tyr                   820      - #           825      - #           830                   - - Val Asp Asn Ile Leu Gly Glu Leu Gly Gly Gl - #u Arg Leu Leu Arg Val               835          - #       840          - #       845                       - - Ala Tyr Gly Asp Glu Met Cys Gly Gln Glu Gl - #n Ser Phe Arg Lys Trp           850              - #   855              - #   860                           - - Ala Pro Glu Val Phe Lys Leu Ala Cys Glu Th - #r Phe Cys Leu Asp Pro       865                 8 - #70                 8 - #75                 8 -       #80                                                                               - - Glu Glu Ser Leu Ser Asp Ala Ser Leu Ala Le - #u Gln Asn Asp Ser         Leu                                                                                              885  - #               890  - #               895              - - Thr Val Asn Thr Val Arg Leu Val Pro Ser Al - #a Asn Lys Gly Ser Leu                   900      - #           905      - #           910                   - - Asp Ser Ser Leu Ser Lys Tyr His Asn Lys Ly - #s Val His Cys Cys Lys               915          - #       920          - #       925                       - - Ala Lys Ala Lys Pro His Asn Leu Thr Arg Le - #u Ser Glu Gly Ala Lys           930              - #   935              - #   940                           - - Thr Thr Met Leu Leu Glu Ile Cys Ala Pro Gl - #y Leu Glu Tyr Glu Pro       945                 9 - #50                 9 - #55                 9 -       #60                                                                               - - Gly Asp His Val Gly Ile Phe Pro Ala Asn Ar - #g Thr Glu Leu Val         Asp                                                                                              965  - #               970  - #               975              - - Gly Leu Leu Asn Arg Leu Val Gly Val Asp As - #n Pro Asp Glu Val Leu                   980      - #           985      - #           990                   - - Gln Leu Gln Leu Leu Lys Glu Lys Gln Thr Se - #r Asn Gly Ile Phe Lys               995          - #       1000          - #      1005                      - - Cys Trp Glu Pro His Asp Lys Ile Pro Pro As - #p Thr Leu Arg Asn Leu           1010             - #   1015              - #  1020                          - - Leu Ala Arg Phe Phe Asp Leu Thr Thr Pro Pr - #o Ser Arg Gln Leu Leu       1025                1030 - #                1035 - #               1040         - - Thr Leu Leu Ala Gly Phe Cys Glu Asp Thr Al - #a Asp Lys Glu Arg Leu                       1045 - #               1050  - #              1055              - - Glu Leu Leu Val Asn Asp Ser Ser Ala Tyr Gl - #u Asp Trp Arg His Trp                   1060     - #           1065      - #          1070                  - - Arg Leu Pro His Leu Leu Asp Val Leu Glu Gl - #u Phe Pro Ser Cys Arg               1075         - #       1080          - #      1085                      - - Pro Pro Ala Pro Leu Leu Leu Ala Gln Leu Th - #r Pro Leu Gln Pro Arg           1090             - #   1095              - #  1100                          - - Phe Tyr Ser Ile Ser Ser Ser Pro Arg Arg Va - #l Ser Asp Glu Ile His       1105                1110 - #                1115 - #               1120         - - Leu Thr Val Ala Ile Val Lys Tyr Arg Cys Gl - #u Asp Gly Gln Gly Asp                       1125 - #               1130  - #              1135              - - Glu Arg Tyr Gly Val Cys Ser Asn Tyr Leu Se - #r Gly Leu Arg Ala Asp                   1140     - #           1145      - #          1150                  - - Asp Glu Leu Phe Met Phe Val Arg Ser Ala Le - #u Gly Phe His Leu Pro               1155         - #       1160          - #      1165                      - - Ser Asp Arg Ser Arg Pro Ile Ile Leu Ile Gl - #y Pro Gly Thr Gly Ile           1170             - #   1175              - #  1180                          - - Ala Pro Phe Arg Ser Phe Trp Gln Glu Phe Gl - #n Val Leu Arg Asp Leu       1185                1190 - #                1195 - #               1200         - - Asp Pro Thr Ala Lys Leu Pro Lys Met Trp Le - #u Phe Phe Gly Cys Arg                       1205 - #               1210  - #              1215              - - Asn Arg Asp Val Asp Leu Tyr Ala Glu Glu Ly - #s Ala Glu Leu Gln Lys                   1220     - #           1225      - #          1230                  - - Asp Gln Ile Leu Asp Arg Val Phe Leu Ala Le - #u Ser Arg Glu Gln Ala               1235         - #       1240          - #      1245                      - - Ile Pro Lys Thr Tyr Val Gln Asp Leu Ile Gl - #u Gln Glu Phe Asp Ser           1250             - #   1255              - #  1260                          - - Leu Tyr Gln Leu Ile Val Gln Glu Arg Gly Hi - #s Ile Tyr Val Cys Gly       1265                1270 - #                1275 - #               1280         - - Asp Val Thr Met Ala Glu His Val Tyr Gln Th - #r Ile Arg Lys Cys Ile                       1285 - #               1290  - #              1295              - - Ala Gly Lys Glu Gln Lys Ser Glu Ala Glu Va - #l Glu Thr Phe Leu Leu                   1300     - #           1305      - #          1310                  - - Thr Leu Arg Asp Glu Ser Arg Tyr His Glu As - #p Ile Phe Gly Ile Thr               1315         - #       1320          - #      1325                      - - Leu Arg Thr Ala Glu Ile His Thr Lys Ser Ar - #g Ala Thr Ala Arg Ile           1330             - #   1335              - #  1340                          - - Arg Met Ala Ser Gln Pro                                                   1345                1350                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1205 amino - #acids                                                (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                               - - Met Gly Asn Leu Lys Ser Val Gly Gln Glu Pr - #o Gly Pro Pro Cys Gly       1               5   - #                10  - #                15                - - Leu Gly Leu Gly Leu Gly Leu Gly Leu Cys Gl - #y Lys Gln Gly Pro Ala                   20      - #            25      - #            30                    - - Ser Pro Ala Pro Glu Pro Ser Arg Ala Pro Al - #a Pro Ala Thr Pro His               35          - #        40          - #        45                        - - Ala Pro Asp His Ser Pro Ala Pro Asn Ser Pr - #o Thr Leu Thr Arg Pro           50              - #    55              - #    60                            - - Pro Glu Gly Pro Lys Phe Pro Arg Val Lys As - #n Trp Glu Leu Gly Ser       65                  - #70                  - #75                  - #80         - - Ile Thr Tyr Asp Thr Leu Cys Ala Gln Ser Gl - #n Gln Asp Gly Pro Cys                       85  - #                90  - #                95                - - Thr Pro Arg Arg Cys Leu Gly Ser Leu Val Le - #u Pro Arg Lys Leu Gln                   100      - #           105      - #           110                   - - Thr Arg Pro Ser Pro Gly Pro Pro Pro Ala Gl - #u Gln Leu Leu Ser Gln               115          - #       120          - #       125                       - - Ala Arg Asp Phe Ile Asn Gln Tyr Tyr Ser Se - #r Ile Lys Arg Ser Gly           130              - #   135              - #   140                           - - Ser Gln Ala His Glu Glu Arg Leu Gln Glu Va - #l Glu Ala Glu Val Ala       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Ser Thr Gly Thr Ile His Leu Arg Glu Ser Gl - #u Leu Val Phe Gly         Ala                                                                                              165  - #               170  - #               175              - - Lys Gln Ala Trp Arg Asn Ala Pro Arg Cys Va - #l Gly Arg Ile Gln Trp                   180      - #           185      - #           190                   - - Gly Lys Leu Gln Val Phe Asp Ala Arg Asp Cy - #s Ser Ser Ala Gln Glu               195          - #       200          - #       205                       - - Met Phe Thr Tyr Ile Cys Asn His Ile Lys Ty - #r Ala Thr Asn Arg Gly           210              - #   215              - #   220                           - - Asn Leu Arg Ser Ala Ile Thr Val Phe Pro Gl - #n Arg Ala Pro Gly Arg       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Gly Asp Phe Arg Ile Trp Asn Ser Gln Leu Va - #l Arg Tyr Ala Gly         Tyr                                                                                              245  - #               250  - #               255              - - Arg Gln Gln Asp Gly Ser Val Arg Gly Asp Pr - #o Ala Asn Val Glu Ile                   260      - #           265      - #           270                   - - Thr Glu Leu Cys Ile Gln His Gly Trp Thr Pr - #o Gly Asn Gly Arg Phe               275          - #       280          - #       285                       - - Asp Val Leu Pro Leu Leu Leu Gln Ala Pro As - #p Glu Ala Pro Glu Leu           290              - #   295              - #   300                           - - Phe Val Leu Pro Pro Glu Leu Val Leu Glu Va - #l Pro Leu Gly Ala Pro       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - His Thr Gly Val Val Arg Gly Pro Gly Leu Ar - #g Trp Tyr Ala Leu         Pro                                                                                              325  - #               330  - #               335              - - Ala Val Ser Asn Met Leu Leu Glu Ile Gly Gl - #y Leu Glu Phe Ser Ala                   340      - #           345      - #           350                   - - Ala Pro Phe Ser Gly Trp Tyr Met Ser Thr Gl - #u Ile Gly Thr Arg Asn               355          - #       360          - #       365                       - - Leu Cys Asp Pro His Arg Tyr Asn Ile Leu Gl - #u Asp Val Ala Val Cys           370              - #   375              - #   380                           - - Met Asp Leu Asp Thr Arg Thr Thr Ser Ser Le - #u Trp Lys Asp Lys Ala       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Ala Val Glu Ile Asn Leu Ala Val Leu His Se - #r Phe Gln Leu Ala         Lys                                                                                              405  - #               410  - #               415              - - Val Thr Ile Val Asp His His Ala Ala Thr Va - #l Ser Phe Met Lys His                   420      - #           425      - #           430                   - - Leu Asp Asn Glu Gln Lys Ala Arg Gly Gly Cy - #s Pro Ala Asp Trp Ala               435          - #       440          - #       445                       - - Trp Ile Val Pro Pro Ile Tyr Gly Ser Leu Pr - #o Pro Val Phe His Gln           450              - #   455              - #   460                           - - Glu Met Val Asn Tyr Ile Leu Ser Pro Ala Ph - #e Arg Tyr Gln Pro Asp       465                 4 - #70                 4 - #75                 4 -       #80                                                                               - - Pro Trp Lys Gly Ser Ala Thr Lys Gly Ala Gl - #y Ile Thr Arg Lys         Lys                                                                                              485  - #               490  - #               495              - - Thr Phe Lys Glu Val Ala Asn Ala Val Lys Il - #e Ser Ala Ser Leu Met                   500      - #           505      - #           510                   - - Gly Thr Leu Met Ala Lys Arg Val Lys Ala Th - #r Ile Leu Tyr Ala Ser               515          - #       520          - #       525                       - - Glu Thr Gly Arg Ala Gln Ser Tyr Ala Gln Gl - #n Leu Gly Arg Leu Phe           530              - #   535              - #   540                           - - Arg Lys Ala Phe Asp Pro Arg Val Leu Cys Me - #t Asp Glu Tyr Asp Val       545                 5 - #50                 5 - #55                 5 -       #60                                                                               - - Val Ser Leu Glu His Glu Ala Leu Val Leu Va - #l Val Thr Ser Thr         Phe                                                                                              565  - #               570  - #               575              - - Gly Asn Gly Asp Pro Pro Glu Asn Gly Glu Se - #r Phe Ala Ala Ala Leu                   580      - #           585      - #           590                   - - Met Glu Met Ser Gly Pro Tyr Asn Ser Ser Pr - #o Arg Pro Glu Gln His               595          - #       600          - #       605                       - - Lys Ser Tyr Lys Ile Arg Phe Asn Ser Val Se - #r Cys Ser Asp Pro Leu           610              - #   615              - #   620                           - - Val Ser Ser Trp Arg Arg Lys Arg Lys Glu Se - #r Ser Asn Thr Asp Ser       625                 6 - #30                 6 - #35                 6 -       #40                                                                               - - Ala Gly Ala Leu Gly Thr Leu Arg Phe Cys Va - #l Phe Gly Leu Gly         Ser                                                                                              645  - #               650  - #               655              - - Arg Ala Tyr Pro His Phe Cys Ala Phe Ala Ar - #g Ala Val Asp Thr Arg                   660      - #           665      - #           670                   - - Leu Glu Glu Leu Gly Gly Glu Arg Leu Leu Gl - #n Leu Gly Gln Gly Asp               675          - #       680          - #       685                       - - Glu Leu Cys Gly Gln Glu Glu Ala Phe Arg Gl - #y Trp Ala Lys Ala Ala           690              - #   695              - #   700                           - - Phe Gln Ala Ser Cys Glu Thr Phe Cys Val Gl - #y Glu Glu Ala Lys Ala       705                 7 - #10                 7 - #15                 7 -       #20                                                                               - - Ala Ala Gln Asp Ile Phe Ser Pro Lys Arg Se - #r Trp Lys Arg Gln         Arg                                                                                              725  - #               730  - #               735              - - Tyr Arg Leu Ser Ala Gln Ala Glu Gly Leu Gl - #n Leu Leu Pro Gly Leu                   740      - #           745      - #           750                   - - Ile His Val His Arg Arg Lys Met Phe Gln Al - #a Thr Val Leu Ser Val               755          - #       760          - #       765                       - - Glu Asn Leu Gln Ser Ser Lys Ser Thr Arg Al - #a Thr Ile Leu Val Arg           770              - #   775              - #   780                           - - Leu Asp Thr Ala Gly Gln Glu Gly Leu Gln Ty - #r Gln Pro Gly Asp His       785                 7 - #90                 7 - #95                 8 -       #00                                                                               - - Ile Gly Ile Ser Ala Pro Asn Arg Pro Gly Le - #u Val Glu Ala Leu         Leu                                                                                              805  - #               810  - #               815              - - Ser Arg Val Glu Asp Pro Pro Pro Pro Thr Gl - #u Ser Val Ala Val Glu                   820      - #           825      - #           830                   - - Gln Leu Glu Lys Gly Ser Pro Gly Gly Pro Pr - #o Pro Ser Trp Val Arg               835          - #       840          - #       845                       - - Asp Pro Arg Leu Pro Pro Cys Thr Val Arg Gl - #n Ala Leu Thr Phe Phe           850              - #   855              - #   860                           - - Leu Asp Ile Thr Ser Pro Pro Ser Pro Arg Le - #u Leu Arg Leu Leu Ser       865                 8 - #70                 8 - #75                 8 -       #80                                                                               - - Thr Leu Ala Glu Glu Pro Ser Glu Gln Gln Gl - #u Leu Glu Thr Leu         Ser                                                                                              885  - #               890  - #               895              - - Gln Asp Pro Arg Arg Tyr Glu Glu Trp Lys Le - #u Val Arg Cys Pro Thr                   900      - #           905      - #           910                   - - Leu Leu Glu Val Leu Glu Gln Phe Pro Ser Va - #l Ala Leu Pro Ala Pro               915          - #       920          - #       925                       - - Leu Leu Leu Thr Gln Leu Pro Leu Leu Gln Pr - #o Arg Tyr Tyr Ser Val           930              - #   935              - #   940                           - - Ser Ser Ala Pro Asn Ala His Pro Gly Glu Va - #l His Leu Thr Val Ala       945                 9 - #50                 9 - #55                 9 -       #60                                                                               - - Val Leu Ala Tyr Arg Thr Gln Asp Gly Leu Gl - #y Pro Leu His Tyr         Gly                                                                                              965  - #               970  - #               975              - - Val Cys Ser Thr Trp Leu Ser Gln Leu Lys Th - #r Gly Asp Pro Val Pro                   980      - #           985      - #           990                   - - Cys Phe Ile Arg Gly Ala Pro Ser Phe Arg Le - #u Pro Pro Asp Pro Tyr               995          - #       1000          - #      1005                      - - Val Pro Cys Ile Leu Val Gly Pro Gly Thr Gl - #y Ile Ala Pro Phe Arg           1010             - #   1015              - #  1020                          - - Gly Phe Trp Gln Glu Arg Leu His Asp Ile Gl - #u Ser Lys Gly Leu Gln       1025                1030 - #                1035 - #               1040         - - Pro His Pro Met Thr Leu Val Phe Gly Cys Ar - #g Cys Ser Gln Leu Asp                       1045 - #               1050  - #              1055              - - His Leu Tyr Arg Asp Glu Val Gln Asp Ala Gl - #n Glu Arg Gly Val Phe                   1060     - #           1065      - #          1070                  - - Gly Arg Val Leu Thr Ala Phe Ser Arg Glu Pr - #o Asp Ser Pro Lys Thr               1075         - #       1080          - #      1085                      - - Tyr Val Gln Asp Ile Leu Arg Thr Glu Leu Al - #a Ala Glu Val His Arg           1090             - #   1095              - #  1100                          - - Val Leu Cys Leu Glu Arg Gly His Met Phe Va - #l Cys Gly Asp Val Thr       1105                1110 - #                1115 - #               1120         - - Met Ala Thr Ser Val Leu Gln Thr Val Gln Ar - #g Ile Leu Ala Thr Glu                       1125 - #               1130  - #              1135              - - Gly Asp Met Glu Leu Asp Glu Ala Gly Asp Va - #l Ile Gly Val Leu Arg                   1140     - #           1145      - #          1150                  - - Asp Gln Gln Arg Tyr His Glu Asp Ile Phe Gl - #y Leu Thr Leu Arg Thr               1155         - #       1160          - #      1165                      - - Gln Glu Val Thr Ser Arg Ile Arg Thr Gln Se - #r Phe Ser Leu Gln Glu           1170             - #   1175              - #  1180                          - - Arg His Leu Arg Gly Ala Val Pro Trp Ala Ph - #e Asp Pro Pro Gly Pro       1185                1190 - #                1195 - #               1200         - - Asp Thr Pro Gly Pro                                                                       1205                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1429 amino - #acids                                                (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                               - - Met Glu Glu Asn Thr Phe Gly Val Gln Gln Il - #e Gln Pro Asn Val Ile       1               5   - #                10  - #                15                - - Ser Val Arg Leu Phe Lys Arg Lys Val Gly Gl - #y Leu Gly Phe Leu Val                   20      - #            25      - #            30                    - - Lys Glu Arg Val Ser Lys Pro Pro Val Ile Il - #e Ser Asp Leu Ile Arg               35          - #        40          - #        45                        - - Gly Gly Ala Ala Glu Gln Ser Gly Leu Ile Gl - #n Ala Gly Asp Ile Ile           50              - #    55              - #    60                            - - Leu Ala Val Asn Asp Arg Pro Leu Val Asp Le - #u Ser Tyr Asp Ser Ala       65                  - #70                  - #75                  - #80         - - Leu Glu Val Leu Arg Gly Ile Ala Ser Glu Th - #r His Val Val Leu Ile                       85  - #                90  - #                95                - - Leu Arg Gly Pro Glu Gly Phe Thr Thr His Le - #u Glu Thr Thr Phe Thr                   100      - #           105      - #           110                   - - Gly Asp Gly Thr Pro Lys Thr Ile Arg Val Th - #r Gln Pro Leu Gly Pro               115          - #       120          - #       125                       - - Pro Thr Lys Ala Val Asp Leu Ser His Gln Pr - #o Ser Ala Ser Lys Asp           130              - #   135              - #   140                           - - Gln Ser Leu Ala Val Asp Arg Val Thr Gly Le - #u Gly Asn Gly Pro Gln       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - His Ala Gln Gly His Gly Gln Gly Ala Gly Se - #r Val Ser Gln Ala         Asn                                                                                              165  - #               170  - #               175              - - Gly Val Ala Ile Asp Pro Thr Met Lys Ser Th - #r Lys Ala Asn Leu Gln                   180      - #           185      - #           190                   - - Asp Ile Gly Glu His Asp Glu Leu Leu Lys Gl - #u Ile Glu Pro Val Leu               195          - #       200          - #       205                       - - Ser Ile Leu Asn Ser Gly Ser Lys Ala Thr As - #n Arg Gly Gly Pro Ala           210              - #   215              - #   220                           - - Lys Ala Glu Met Lys Asp Thr Gly Ile Gln Va - #l Asp Arg Asp Leu Asp       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Gly Lys Ser His Lys Ala Pro Pro Leu Gly Gl - #y Asp Asn Asp Arg         Val                                                                                              245  - #               250  - #               255              - - Phe Asn Asp Leu Trp Gly Lys Asp Asn Val Pr - #o Val Ile Leu Asn Asn                   260      - #           265      - #           270                   - - Pro Tyr Ser Glu Lys Glu Gln Ser Pro Thr Se - #r Gly Lys Gln Ser Pro               275          - #       280          - #       285                       - - Thr Lys Asn Gly Ser Pro Ser Arg Cys Pro Ar - #g Phe Leu Lys Val Lys           290              - #   295              - #   300                           - - Asn Trp Glu Thr Asp Val Val Leu Thr Asp Th - #r Leu His Leu Lys Ser       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Thr Leu Glu Thr Gly Cys Thr Glu His Ile Cy - #s Met Gly Ser Ile         Met                                                                                              325  - #               330  - #               335              - - Leu Pro Ser Gln His Thr Arg Lys Pro Glu As - #p Val Arg Thr Lys Asp                   340      - #           345      - #           350                   - - Gln Leu Phe Pro Leu Ala Lys Glu Phe Leu As - #p Gln Tyr Tyr Ser Ser               355          - #       360          - #       365                       - - Ile Lys Arg Phe Gly Ser Lys Ala His Met As - #p Arg Leu Glu Glu Val           370              - #   375              - #   380                           - - Asn Lys Glu Ile Glu Ser Thr Ser Thr Tyr Gl - #n Leu Lys Asp Thr Glu       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Leu Ile Tyr Gly Ala Lys His Ala Trp Arg As - #n Ala Ser Arg Cys         Val                                                                                              405  - #               410  - #               415              - - Gly Arg Ile Gln Trp Ser Lys Leu Gln Val Ph - #e Asp Ala Arg Asp Cys                   420      - #           425      - #           430                   - - Thr Thr Ala His Gly Met Phe Asn Tyr Ile Cy - #s Asn His Val Lys Tyr               435          - #       440          - #       445                       - - Ala Thr Asn Lys Gly Asn Leu Arg Ser Ala Il - #e Thr Ile Phe Pro Gln           450              - #   455              - #   460                           - - Arg Thr Asp Gly Lys His Asp Phe Arg Val Tr - #p Asn Ser Gln Leu Ile       465                 4 - #70                 4 - #75                 4 -       #80                                                                               - - Arg Tyr Ala Gly Tyr Lys Gln Pro Asp Gly Se - #r Thr Leu Gly Asp         Pro                                                                                              485  - #               490  - #               495              - - Ala Asn Val Gln Phe Thr Glu Ile Cys Ile Gl - #n Gln Gly Trp Lys Ala                   500      - #           505      - #           510                   - - Pro Arg Gly Arg Phe Asp Val Leu Pro Leu Le - #u Leu Gln Ala Asn Gly               515          - #       520          - #       525                       - - Asn Asp Pro Glu Leu Phe Gln Ile Pro Pro Gl - #u Leu Val Leu Glu Val           530              - #   535              - #   540                           - - Pro Ile Arg His Pro Lys Phe Asp Trp Phe Ly - #s Asp Leu Gly Leu Lys       545                 5 - #50                 5 - #55                 5 -       #60                                                                               - - Trp Tyr Gly Leu Pro Ala Val Ser Asn Met Le - #u Leu Glu Ile Gly         Gly                                                                                              565  - #               570  - #               575              - - Leu Glu Phe Ser Ala Cys Pro Phe Ser Gly Tr - #p Tyr Met Gly Thr Glu                   580      - #           585      - #           590                   - - Ile Gly Val Arg Asp Tyr Cys Asp Asn Ser Ar - #g Tyr Asn Ile Leu Glu               595          - #       600          - #       605                       - - Glu Val Ala Lys Lys Met Asp Leu Asp Met Ar - #g Lys Thr Ser Ser Leu           610              - #   615              - #   620                           - - Trp Lys Asp Gln Ala Leu Val Glu Ile Asn Il - #e Ala Val Leu Tyr Ser       625                 6 - #30                 6 - #35                 6 -       #40                                                                               - - Phe Gln Ser Asp Lys Val Thr Ile Val Asp Hi - #s His Ser Ala Thr         Glu                                                                                              645  - #               650  - #               655              - - Ser Phe Ile Lys His Met Glu Asn Glu Tyr Ar - #g Cys Arg Gly Gly Cys                   660      - #           665      - #           670                   - - Pro Ala Asp Trp Val Trp Ile Val Pro Pro Me - #t Ser Gly Ser Ile Thr               675          - #       680          - #       685                       - - Pro Val Phe His Gln Glu Met Leu Asn Tyr Ar - #g Leu Thr Pro Ser Phe           690              - #   695              - #   700                           - - Glu Tyr Gln Pro Asp Pro Trp Asn Thr His Va - #l Trp Lys Gly Thr Asn       705                 7 - #10                 7 - #15                 7 -       #20                                                                               - - Gly Thr Pro Thr Lys Arg Arg Ala Ile Gly Ph - #e Lys Lys Leu Ala         Glu                                                                                              725  - #               730  - #               735              - - Ala Val Lys Phe Ser Ala Lys Leu Met Gly Gl - #n Ala Met Ala Lys Arg                   740      - #           745      - #           750                   - - Val Lys Ala Thr Ile Leu Tyr Ala Thr Glu Th - #r Gly Lys Ser Gln Ala               755          - #       760          - #       765                       - - Tyr Ala Lys Thr Leu Cys Glu Ile Phe Lys Hi - #s Ala Phe Asp Ala Lys           770              - #   775              - #   780                           - - Ala Met Ser Met Glu Glu Tyr Asp Ile Val Hi - #s Leu Glu His Glu Ala       785                 7 - #90                 7 - #95                 8 -       #00                                                                               - - Leu Val Leu Val Val Thr Ser Thr Phe Gly As - #n Gly Asp Pro Pro         Glu                                                                                              805  - #               810  - #               815              - - Asn Gly Glu Lys Phe Gly Cys Ala Leu Met Gl - #u Met Arg His Pro Asn                   820      - #           825      - #           830                   - - Ser Val Gln Glu Glu Arg Lys Ser Tyr Lys Va - #l Arg Phe Asn Ser Val               835          - #       840          - #       845                       - - Ser Ser Tyr Ser Asp Ser Arg Lys Ser Ser Gl - #y Asp Gly Pro Asp Leu           850              - #   855              - #   860                           - - Arg Asp Asn Phe Glu Ser Thr Gly Pro Leu Al - #a Asn Val Arg Phe Ser       865                 8 - #70                 8 - #75                 8 -       #80                                                                               - - Val Phe Gly Leu Gly Ser Arg Ala Tyr Pro Hi - #s Phe Cys Ala Phe         Gly                                                                                              885  - #               890  - #               895              - - His Ala Val Asp Thr Leu Leu Glu Glu Leu Gl - #y Gly Glu Arg Ile Leu                   900      - #           905      - #           910                   - - Lys Met Arg Glu Gly Asp Glu Leu Cys Gly Gl - #n Glu Glu Ala Phe Arg               915          - #       920          - #       925                       - - Thr Trp Ala Lys Lys Val Phe Lys Ala Ala Cy - #s Asp Val Phe Cys Val           930              - #   935              - #   940                           - - Gly Asp Asp Val Asn Ile Glu Lys Pro Asn As - #n Ser Leu Ile Ser Asn       945                 9 - #50                 9 - #55                 9 -       #60                                                                               - - Asp Arg Ser Trp Lys Arg Asn Lys Phe Arg Le - #u Thr Tyr Val Ala         Glu                                                                                              965  - #               970  - #               975              - - Ala Pro Asp Leu Thr Gln Gly Leu Ser Asn Va - #l His Lys Lys Arg Val                   980      - #           985      - #           990                   - - Ser Ala Ala Arg Leu Leu Ser Arg Gln Asn Le - #u Gln Ser Pro Lys Phe               995          - #       1000          - #      1005                      - - Ser Arg Ser Thr Ile Phe Val Arg Leu His Th - #r Asn Gly Asn Gln Glu           1010             - #   1015              - #  1020                          - - Leu Gln Tyr Gln Pro Gly Asp His Leu Gly Va - #l Phe Pro Gly Asn His       1025                1030 - #                1035 - #               1040         - - Glu Asp Leu Val Asn Ala Leu Ile Glu Arg Le - #u Glu Asp Ala Pro Pro                       1045 - #               1050  - #              1055              - - Ala Asn His Val Val Lys Val Glu Met Leu Gl - #u Glu Arg Asn Thr Ala                   1060     - #           1065      - #          1070                  - - Leu Gly Val Ile Ser Asn Trp Lys Asp Glu Se - #r Arg Leu Pro Pro Cys               1075         - #       1080          - #      1085                      - - Thr Ile Phe Gln Ala Phe Lys Tyr Tyr Leu As - #p Ile Thr Thr Pro Pro           1090             - #   1095              - #  1100                          - - Thr Pro Leu Gln Leu Gln Gln Phe Ala Ser Le - #u Ala Thr Asn Glu Lys       1105                1110 - #                1115 - #               1120         - - Glu Lys Gln Arg Leu Leu Val Leu Ser Lys Gl - #y Leu Gln Glu Tyr Glu                       1125 - #               1130  - #              1135              - - Glu Trp Lys Trp Gly Lys Asn Pro Thr Met Va - #l Glu Val Leu Glu Glu                   1140     - #           1145      - #          1150                  - - Phe Pro Ser Ile Gln Met Pro Ala Thr Leu Le - #u Leu Thr Gln Leu Ser               1155         - #       1160          - #      1165                      - - Leu Leu Gln Pro Arg Tyr Tyr Ser Ile Ser Se - #r Ser Pro Asp Met Tyr           1170             - #   1175              - #  1180                          - - Pro Asp Glu Val His Leu Thr Val Ala Ile Va - #l Ser Tyr His Thr Arg       1185                1190 - #                1195 - #               1200         - - Asp Gly Glu Gly Pro Val His His Gly Val Cy - #s Ser Ser Trp Leu Asn                       1205 - #               1210  - #              1215              - - Arg Ile Gln Ala Asp Asp Val Val Pro Cys Ph - #e Val Arg Gly Ala Pro                   1220     - #           1225      - #          1230                  - - Ser Phe His Leu Pro Arg Asn Pro Gln Val Pr - #o Cys Ile Leu Val Gly               1235         - #       1240          - #      1245                      - - Pro Gly Thr Gly Ile Ala Pro Phe Arg Ser Ph - #e Trp Gln Gln Arg Gln           1250             - #   1255              - #  1260                          - - Phe Asp Ile Gln His Lys Gly Met Asn Pro Cy - #s Pro Met Val Leu Val       1265                1270 - #                1275 - #               1280         - - Phe Gly Cys Arg Gln Ser Lys Ile Asp His Il - #e Tyr Arg Glu Glu Thr                       1285 - #               1290  - #              1295              - - Leu Gln Ala Lys Asn Lys Gly Val Phe Arg Gl - #u Leu Tyr Thr Ala Tyr                   1300     - #           1305      - #          1310                  - - Ser Arg Glu Pro Asp Arg Pro Lys Lys Tyr Va - #l Gln Asp Val Leu Gln               1315         - #       1320          - #      1325                      - - Glu Gln Leu Ala Glu Ser Val Tyr Arg Ala Le - #u Lys Glu Gln Gly Gly           1330             - #   1335              - #  1340                          - - His Ile Tyr Val Cys Gly Asp Val Thr Met Al - #a Ala Asp Val Leu Lys       1345                1350 - #                1355 - #               1360         - - Ala Ile Gln Arg Ile Met Thr Gln Gln Gly Ly - #s Leu Ser Glu Glu Asp                       1365 - #               1370  - #              1375              - - Ala Gly Val Phe Ile Ser Arg Leu Arg Asp As - #p Asn Arg Tyr His Glu                   1380     - #           1385      - #          1390                  - - Asp Ile Phe Gly Val Thr Leu Arg Thr Tyr Gl - #u Val Thr Asn Arg Leu               1395         - #       1400          - #      1405                      - - Arg Ser Glu Ser Ile Ala Phe Ile Glu Glu Se - #r Lys Lys Asp Ala Asp           1410             - #   1415              - #  1420                          - - Glu Val Phe Ser Ser                                                       1425                                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1144 amino - #acids                                                (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                               - - Met Ala Cys Pro Trp Lys Phe Leu Phe Lys Va - #l Lys Ser Tyr Gln Ser       1               5   - #                10  - #                15                - - Asp Leu Lys Glu Glu Lys Asp Ile Asn Asn As - #n Val Lys Lys Thr Pro                   20      - #            25      - #            30                    - - Cys Ala Val Leu Ser Pro Thr Ile Gln Asp As - #p Pro Lys Ser His Gln               35          - #        40          - #        45                        - - Asn Gly Ser Pro Gln Leu Leu Thr Gly Thr Al - #a Gln Asn Val Pro Glu           50              - #    55              - #    60                            - - Ser Leu Asp Lys Leu His Val Thr Ser Thr Ar - #g Pro Gln Tyr Val Arg       65                  - #70                  - #75                  - #80         - - Ile Lys Asn Trp Gly Ser Gly Glu Ile Leu Hi - #s Asp Thr Leu His His                       85  - #                90  - #                95                - - Lys Ala Thr Ser Asp Phe Thr Cys Lys Ser Ly - #s Ser Cys Leu Gly Ser                   100      - #           105      - #           110                   - - Ile Met Asn Pro Lys Ser Leu Thr Arg Gly Pr - #o Arg Asp Lys Pro Thr               115          - #       120          - #       125                       - - Pro Leu Glu Glu Leu Leu Pro His Ala Ile Gl - #u Phe Ile Asn Gln Tyr           130              - #   135              - #   140                           - - Tyr Gly Ser Phe Lys Glu Ala Lys Ile Glu Gl - #u His Leu Ala Arg Leu       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Glu Ala Val Thr Lys Glu Ile Glu Thr Thr Gl - #y Thr Tyr Gln Leu         Thr                                                                                              165  - #               170  - #               175              - - Leu Asp Glu Leu Ile Phe Ala Thr Lys Met Al - #a Trp Arg Asn Ala Pro                   180      - #           185      - #           190                   - - Arg Cys Ile Gly Arg Ile Gln Trp Ser Asn Le - #u Gln Val Phe Asp Ala               195          - #       200          - #       205                       - - Arg Asn Cys Ser Thr Ala Gln Glu Met Phe Gl - #n His Ile Cys Arg His           210              - #   215              - #   220                           - - Ile Leu Tyr Ala Thr Asn Asn Gly Asn Ile Ar - #g Ser Ala Ile Thr Val       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Phe Pro Gln Arg Ser Asp Gly Lys His Asp Ph - #e Arg Leu Trp Asn         Ser                                                                                              245  - #               250  - #               255              - - Gln Leu Ile Arg Tyr Ala Gly Tyr Gln Met Pr - #o Asp Gly Thr Ile Arg                   260      - #           265      - #           270                   - - Gly Asp Ala Ala Thr Leu Glu Phe Thr Gln Le - #u Cys Ile Asp Leu Gly               275          - #       280          - #       285                       - - Trp Lys Pro Arg Tyr Gly Arg Phe Asp Val Le - #u Pro Leu Val Leu Gln           290              - #   295              - #   300                           - - Ala Asp Gly Gln Asp Pro Glu Val Phe Glu Il - #e Pro Pro Asp Leu Val       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Leu Glu Val Thr Met Glu His Pro Lys Tyr Gl - #u Trp Phe Gln Glu         Leu                                                                                              325  - #               330  - #               335              - - Gly Leu Lys Trp Tyr Ala Leu Pro Ala Val Al - #a Asn Met Leu Leu Glu                   340      - #           345      - #           350                   - - Val Gly Gly Leu Glu Phe Pro Ala Cys Pro Ph - #e Asn Gly Trp Tyr Met               355          - #       360          - #       365                       - - Gly Thr Glu Ile Gly Val Arg Asp Phe Cys As - #p Thr Gln Arg Tyr Asn           370              - #   375              - #   380                           - - Ile Leu Glu Glu Val Gly Arg Arg Met Gly Le - #u Glu Thr His Thr Leu       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Ala Ser Leu Trp Lys Asp Arg Ala Val Thr Gl - #u Ile Asn Val Ala         Val                                                                                              405  - #               410  - #               415              - - Leu His Ser Phe Gln Lys Gln Asn Val Thr Il - #e Met Asp His His Thr                   420      - #           425      - #           430                   - - Ala Ser Glu Ser Phe Met Lys His Met Gln As - #n Glu Tyr Arg Ala Arg               435          - #       440          - #       445                       - - Gly Gly Cys Pro Ala Asp Trp Ile Trp Leu Va - #l Pro Pro Val Ser Gly           450              - #   455              - #   460                           - - Ser Ile Thr Pro Val Phe His Gln Glu Met Le - #u Asn Tyr Val Leu Ser       465                 4 - #70                 4 - #75                 4 -       #80                                                                               - - Pro Phe Tyr Tyr Tyr Gln Ile Glu Pro Trp Ly - #s Thr His Ile Trp         Gln                                                                                              485  - #               490  - #               495              - - Asn Glu Lys Leu Arg Pro Arg Arg Arg Glu Il - #e Arg Phe Arg Val Leu                   500      - #           505      - #           510                   - - Val Lys Val Val Phe Phe Ala Ser Met Leu Me - #t Arg Lys Val Met Ala               515          - #       520          - #       525                       - - Ser Arg Val Arg Ala Thr Val Leu Phe Ala Th - #r Glu Thr Gly Lys Ser           530              - #   535              - #   540                           - - Glu Ala Leu Ala Arg Asp Leu Ala Thr Leu Ph - #e Ser Tyr Ala Phe Asn       545                 5 - #50                 5 - #55                 5 -       #60                                                                               - - Thr Lys Val Val Cys Met Asp Gln Tyr Lys Al - #a Ser Thr Leu Glu         Glu                                                                                              565  - #               570  - #               575              - - Glu Gln Leu Leu Leu Val Val Thr Ser Thr Ph - #e Gly Asn Gly Asp Cys                   580      - #           585      - #           590                   - - Pro Ser Asn Gly Gln Thr Leu Lys Lys Ser Le - #u Phe Met Leu Arg Glu               595          - #       600          - #       605                       - - Leu Asn His Thr Phe Arg Tyr Ala Val Phe Gl - #y Leu Gly Ser Ser Met           610              - #   615              - #   620                           - - Tyr Pro Gln Phe Cys Ala Phe Ala His Asp Il - #e Asp Gln Lys Leu Ser       625                 6 - #30                 6 - #35                 6 -       #40                                                                               - - His Leu Gly Ala Ser Gln Leu Ala Pro Thr Gl - #y Glu Gly Asp Glu         Leu                                                                                              645  - #               650  - #               655              - - Ser Gly Gln Glu Asp Ala Phe Arg Ser Trp Al - #a Val Gln Thr Phe Arg                   660      - #           665      - #           670                   - - Ala Ala Cys Glu Thr Phe Asp Val Arg Ser Ly - #s His His Ile Gln Ile               675          - #       680          - #       685                       - - Pro Lys Arg Phe Thr Ser Asn Ala Thr Trp Gl - #u Pro Gln Gln Tyr Arg           690              - #   695              - #   700                           - - Leu Ile Gln Ser Pro Glu Pro Leu Asp Leu As - #n Arg Ala Leu Ser Ser       705                 7 - #10                 7 - #15                 7 -       #20                                                                               - - Ile His Ala Lys Asn Val Phe Thr Met Arg Le - #u Lys Ser Gln Gln         Asn                                                                                              725  - #               730  - #               735              - - Leu Gln Ser Glu Lys Ser Ser Arg Thr Thr Le - #u Leu Val Gln Leu Thr                   740      - #           745      - #           750                   - - Phe Glu Gly Ser Arg Gly Pro Ser Tyr Leu Pr - #o Gly Glu His Leu Gly               755          - #       760          - #       765                       - - Ile Phe Pro Gly Asn Gln Thr Ala Leu Val Gl - #n Gly Ile Leu Glu Arg           770              - #   775              - #   780                           - - Val Val Asp Cys Pro Thr Pro His Gln Thr Va - #l Cys Leu Glu Val Leu       785                 7 - #90                 7 - #95                 8 -       #00                                                                               - - Asp Glu Ser Gly Ser Tyr Trp Val Lys Asp Ly - #s Arg Leu Pro Pro         Cys                                                                                              805  - #               810  - #               815              - - Ser Leu Ser Gln Ala Leu Thr Tyr Phe Leu As - #p Ile Thr Thr Pro Pro                   820      - #           825      - #           830                   - - Thr Gln Leu Gln Leu His Lys Leu Ala Arg Ph - #e Ala Thr Asp Glu Thr               835          - #       840          - #       845                       - - Asp Arg Gln Arg Leu Glu Ala Leu Cys Gln Pr - #o Ser Glu Tyr Asn Asp           850              - #   855              - #   860                           - - Trp Lys Phe Ser Asn Asn Pro Thr Phe Leu Gl - #u Val Leu Glu Glu Phe       865                 8 - #70                 8 - #75                 8 -       #80                                                                               - - Pro Ser Leu His Val Pro Ala Ala Phe Leu Le - #u Ser Gln Leu Pro         Ile                                                                                              885  - #               890  - #               895              - - Leu Lys Pro Arg Tyr Tyr Ser Ile Ser Ser Se - #r Gln Asp His Thr Pro                   900      - #           905      - #           910                   - - Ser Glu Val His Leu Thr Val Ala Val Val Th - #r Tyr Arg Thr Arg Asp               915          - #       920          - #       925                       - - Gly Gln Gly Pro Leu His His Gly Val Cys Se - #r Thr Trp Ile Arg Asn           930              - #   935              - #   940                           - - Leu Lys Pro Gln Asp Pro Val Pro Cys Phe Va - #l Arg Ser Val Ser Gly       945                 9 - #50                 9 - #55                 9 -       #60                                                                               - - Phe Gln Leu Pro Glu Asp Pro Ser Gln Pro Cy - #s Ile Leu Ile Gly         Pro                                                                                              965  - #               970  - #               975              - - Gly Thr Gly Ile Ala Pro Phe Arg Ser Phe Tr - #p Gln Gln Arg Leu His                   980      - #           985      - #           990                   - - Asp Ser Gln His Lys Gly Leu Lys Gly Gly Ar - #g Met Ser Leu Val Phe               995          - #       1000          - #      1005                      - - Gly Cys Arg His Pro Glu Glu Asp His Leu Ty - #r Gln Glu Glu Met Gln           1010             - #   1015              - #  1020                          - - Glu Met Val Arg Lys Arg Val Leu Phe Gln Va - #l His Thr Gly Tyr Ser       1025                1030 - #                1035 - #               1040         - - Arg Leu Pro Gly Lys Pro Lys Val Tyr Val Gl - #n Asp Ile Leu Gln Lys                       1045 - #               1050  - #              1055              - - Gln Leu Ala Asn Glu Val Leu Ser Val Leu Hi - #s Gly Glu Gln Gly His                   1060     - #           1065      - #          1070                  - - Leu Tyr Ile Cys Gly Asp Val Arg Met Ala Ar - #g Asp Val Ala Thr Thr               1075         - #       1080          - #      1085                      - - Leu Lys Lys Leu Val Ala Thr Lys Leu Asn Le - #u Ser Glu Glu Gln Val           1090             - #   1095              - #  1100                          - - Glu Asp Tyr Phe Phe Gln Leu Lys Ser Gln Ly - #s Arg Tyr His Glu Asp       1105                1110 - #                1115 - #               1120         - - Ile Phe Gly Ala Val Phe Ser Tyr Gly Ala Ly - #s Lys Gly Ser Ala Leu                       1125 - #               1130  - #              1135              - - Glu Glu Pro Lys Ala Thr Arg Leu                                                       1140                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 20 amino - #acids                                                  (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                               - - Gly Asp Pro Ala Asn Val Glu Phe Thr Glu Il - #e Cys Ile Gln Gln Gly       1               5   - #                10  - #                15                - - Trp Lys Pro Arg                                                                       20                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 20 amino - #acids                                                  (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                               - - Gly Asp Pro Met Asn Val Glu Phe Thr Glu Th - #r Val Ala Leu Lys Met       1               5   - #                10  - #                15                - - Gln Leu Asp Thr                                                                       20                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 20 amino - #acids                                                  (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                               - - Cys Asp Asn Ser Arg Tyr Asn Ile Leu Glu Gl - #u Val Ala Lys Lys Met       1               5   - #                10  - #                15                - - Asp Leu Asp Met                                                                       20                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 20 amino - #acids                                                  (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                               - - Gly Asp Pro Ala Asn Val Glu Phe Thr Glu Gl - #u Val Ala Lys Lys Met       1               5   - #                10  - #                15                - - Asp Leu Asp Met                                                                       20                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 57 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: DNA (genomic)                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                               - - CGTCTAGATC TATGACTGAA TATGACGTAA TATGACGTAA TGGTACCAGA TC - #TGGCC             57                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 41 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: DNA (genomic)                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                               - - AAATGACGTA ACGGAAATGA CGTAACGGAA ATGACGTAAC G    - #                       - #   41                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 42 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: DNA (genomic)                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                               - - AAATGAATTA ACGGAAATGA ATTAACGGAA ATGAATTAAC GG    - #                       - #  42                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 81 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: DNA (genomic)                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                               - - TGCACGGGTT TTCGACGTTC ACTGGTAGTG TCTGATGAGG CCGAAAGGCC GA -              #AACGCGAT     60                                                                  - - GCCCATAACC ACCACGCTCA G           - #                  - #                       - #81                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 100 base - #pairs                                                  (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: DNA (genomic)                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                               - - TCGACCCACA GTTTCGGGTT TTCGAGCAAG TCTGCTAGTG TCTGATGAGG CC -              #GAAAGGCC     60                                                                  - - GAAACGCGAA GCCGTATTGC ACCACGCTCA TCGAGAAGGC     - #                       - #   100                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 55 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: DNA (genomic)                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                               - - CTAGAGCTTG CAAGCATGCT TGCAAGCAAG CATGCTTGCA AGCATGCTTG CA - #AGC               55                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: DNA (genomic)                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                               - - CTCTAGAGCG TACGCAAGCG TACGCAAGCG TACG       - #                  -       #        34                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 17 amino - #acids                                                  (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                               - - Arg Lys Arg Glu Ile Arg Leu Gln Lys Asn Ar - #g Glu Ala Ala Arg         Glu                                                                              1               5   - #                10  - #                15               - - Cys                                                                      __________________________________________________________________________ 

What is claimed is:
 1. A method of potentiating long term memory in an animal comprising treating said animal to increase the level of a homodimer of an endogenous activator relative to the level of said homodimer present in said animal prior to said treatment, wherein said activator is a CREB/CREM/ATF-1 subfamily member.
 2. The method of claim 1 wherein said animal is Drosophila and the activator homodimer is a dCREB2a homodimer.
 3. The method of claim 1 wherein said animal is a mammal.
 4. A method of potentiating long term memory in an animal comprising treating said animal to decrease the level of a heterodimer of an endogenous activator and an endogenous repressor relative to the level of said heterodimer present in said animal prior to said treatment, wherein said activator is a CREB/CREM/ATF-1 subfamily member and said repressor is an antagonist of said activator.
 5. The method of claim 4 wherein said animal is Drosophila and the activator-repressor heterodimer is a dCREB2a-dCRB2b heterodimer.
 6. The method of claim 3 wherein said animal is a mammal.
 7. A method of potentiating long term memory in an animal comprising treating said animal to decrease the level of a homodimer of an endogenous repressor relative to the level of said homodimer present in said animal prior to said treatment, wherein said repressor is an antagonist of a CREB/CREM/ATF-1 subfamily member.
 8. The method of claim 7 wherein said animal is Drosophila and the repressor homodimer is a dCREB2b homodimer.
 9. The method of claim 5 wherein the animal is a mammal.
 10. A method of modulating long term memory in an animal comprising treating said animal to modulate the expression of an endogenous gene, said gene selected from the group consisting of a gene encoding an activator and a gene encoding an antagonist of said activator, wherein said activator is a CREB/CREM/ATF-1 subfamily member.
 11. The method of claim 10 wherein said animal is a mammal and the gene is selected from the group consisting of mammalian CREB and mammalian CREM genes.
 12. The method of claim 10 wherein the gene encodes said activator and inducing said gene results in the potentiation of long term memory.
 13. The method of claim 10 wherein the gene encodes said antagonist and inducing said gene results in the blocking of long term memory.
 14. The method of claim 10 wherein said animal is Drosophila and the gene is selected from the group consisting of dCREB2 and dCREB1 genes.
 15. A method of modulating long term memory in an animal comprising treating said animal to modulate the level of an endogenous activator and the level of an endogenous antagonist of said activator, wherein said activator is a CREB/CREM/ATF-1 subfamily member, and wherein long term memory is potentiated in the animal when the net amount of functional activator (ΔC) is greater than zero.
 16. The method of claim 12 wherein the animal is a mammal.
 17. The method of claim 12 wherein the animal is Drosophila. 